Salts of peptides with carboxy-terminated polyesters

ABSTRACT

This invention relates to novel salts composed of a cation derived from a peptide containing at least one basic group and an anion derived from a carboxy-terminated polyester, processes for the manufacture of such salts, and the use of such salts in the manufacture of extended release pharmaceutical compositions. The salts of the invention possess a variety of properties which are useful in the formulation of extended release pharmaceutical compositions, whether the salts are in pure form or are in admixture with either an excess of the peptide in its free, unbound form or an excess of the free polyester.

[0001] This invention relates to novel salts composed of a cationderived from a peptide containing at least one basic group and an anionderived from a carboxy-terminated polyester, processes for themanufacture of such salts, and the use of such salts in the manufactureof extended release pharmaceutical compositions. The salts of theinvention possess a variety of properties which are useful in theformulation of extended release pharmaceutical compositions, whether thesalts are in pure form or are in admixture with either an excess of thepeptide in its free, unbound form or an excess of the free polyester.Such salts are amphipathic, being comprised in part of a peptide, whichis hydrophilic and lipophobic, and in part a polyester, which ishydrophobic and lipophilic.

[0002] The word “peptide” is used herein in a generic sense to includepoly(amino acids) which are normally generally referred to as“peptides”, “polypeptides” or “proteins”; and a “basic peptide” is apeptide which is basic in nature, arising from the presence of an excessof basic amino acids, for example arginine or lysine, or arising fromthe N-terminus of the peptide, or simply a peptide which contains atleast one basic group, optionally in the presence of one or more acidicamino acid groups. The term also includes synthetic analogues ofpeptides, unnatural amino acids having basic functionality, or any otherform of introduced basicity. The word “polyester” is used hereinafter tomean a carboxy-terminated polyester.

[0003] European Patent No. 58,481 alludes to the possibility of specificchemical interactions between the terminal carboxylic acid group of apolyester and a basic group or groups within a peptide. Lawter et al.,Proc. Int. Symp. Control Rel. Bioact. Mater., 14, 19, (1987) and Okadaet al., Pharmaceutical Research, 8, 584-587 (1991), also refer to thispossibility, but these publications are speculative in this regard, inthat they do not particularly describe any such specificpeptide-polyester salt, do not give any indication of how such salts canbe prepared, and are silent with regard to any beneficial effects whichcould arise from the use of such salts in the manufacture ofpharmaceutical compositions.

[0004] According to the present invention, however, there is provided acomposition containing or comprising, as initially made, a salt formedfrom a cation derived from a peptide containing at least one basic groupand an anion derived from a carboxy-terminated polyester; thecomposition being in the form of a solution or dispersion of the salt ina solvent which is a solvent for the free polyester but not a solventfor the free peptide, the particle size of the salt in said dispersionbeing less than 5 μm and preferably less than 0.2 μm; or in the form ofmicroparticles or an implant for injection or sub-dermal implantation.

[0005] The cation component of the salt may be derived from a basicpeptide which is pharmacologically active, or from a basic peptide whichis pharmacologically inactive. When the basic peptide ispharmacologically active, the salt of the invention itself may beformulated into an extended release pharmaceutical formulation. When thebasic peptide is pharmacologically inactive, the salt of the inventionmay be used as an excipient in the formulation of extended releasecompositions of other, pharmacologically active, peptides which eitherare acidic in nature, (comprising an excess of acidic amino acids suchas aspartic acid and glutamic acid), or are neutral in nature.

[0006] In extended release formulations of peptides, a furtherrequirement, of course, is that the peptide should be substantiallystable in the formulation over the period of release envisaged. By“substantially stable” it is meant that the drug is not rendered totallyinsoluble or denatured, with total loss of pharmacological activity,during the period of use envisaged for the formulation.

[0007] Suitable pharmacologically active peptides have a molecularweight of at least 300 Da, and preferably at least 800 Da. Examples ofsuch peptides which may be substantially stable in the extended releaseformulations over the intended period of release, and which maytherefore be used in the compositions of this invention, are oxytocin,vasopressin, adrenocorticotrophic hormone (ACTH), epidermal growthfactor (EGF), prolactin, luteinising hormone, follicle stimulatinghormone, luliberin or luteinizing hormone releasing hormone (LHRH),insulin, somatostatin, glucagon, interferon, gastrin, tetragastrin,pentagastrin, urogastrone, secretin, calcitonin, enkephalins,endorphins, kyotorphin, taftsin, thymopoietin, thymosin, thymostimulin,thymic humoral factor, serum thymic factor, tumour necrosis factor,colony stimulating factors, motilin, bombesin, dinorphin, neurotensin,cerulein, bradykinin, urokinase, kallikrein, substance P analogues andantagonists, angiotensin II, nerve growth factor, blood coagulationfactor VII and IX, lysozyme chloride, renin, bradykinin, tyrocidin,gramicidines, growth hormones, melanocyte stimulating hormone, thyroidhormone releasing hormone, thyroid stimulating hormone, parathyroidhormone, pancreozymin, cholecystokinin, human placental lactogen, humanchorionic gonadotrophin, protein synthesis stimulating peptide, gastricinhibitory peptide, vasoactive intestinal peptide, platelet derivedgrowth factor, growth hormone releasing factor, bone morphogenicprotein, and synthetic analogues and modifications andpharmacologically-active fragments thereof.

[0008] Preferred peptide components of the compositions of the inventionare synthetic analogues of LHRH, and particular such analogues include,but are not limited to, buserelin ([D-Ser(Bu^(t))⁶, des-Gly-NH₂¹⁰]-LHRH(1-9)NHEt), deslorelin ([D-Trp⁶, des-Gly-NH₂ ¹⁰]-LHRH(1-9)NHEt),fertirelin ([des-Gly-NH₂ ¹⁰]-LHRH(1-9)NHEt), goserelin ([D-Ser(Bu^(t))⁶,Azgly¹⁰]-LHRH), histrelin ([D-His(Bzl)⁶, des-Gly-NH₂ ]-LHRH(1-9)NHEt),leuprorelin ([D-Leu⁶, des-Gly-NH₂ ¹⁰ ]-LHRH(1-9)NHEt), lutrelin ([D-Trp6, HeLeu⁷, des-Gly-NH₂ ¹⁰]-LHRH(1-9)NHEt), nafarelin ([D-Nal]-LHRH),tryptorelin ([D-Trp⁶]-LHRH), and pharmacologically active salts thereof.

[0009] Suitable pharmacologically inactive basic peptides, which may beused in the salts of the invention, are polyarginine, polylysine andpoly(arginine-co-lysine), (co-)polymers of neutral amino acids, in D-,L- or DL-form, with arginine and/or lysine in D-, L- or racemic form, orpeptides or (co-)polypeptides in which the peptide chains are terminatedin whole or in part by a basic group at the N-terminus and the backboneis comprised of neutral amino acid residues.

[0010] The carboxy-terminated polyester used as the source of the anionin the salt of the invention may be a homo-polyester or a co-polyester.Preferred such polyesters are those which degrade or erode in an aqueousphysiological environment, such as that found in intramuscular orsubcutaneous tissue, to low molecular weight water-soluble fragments. Inthis environment, the dominant process of degradation is simple bulkhydrolysis, involving hydrolytic scission of ester groups, which leadsto lower molecular weight homo- or co-polyester fragments, andultimately to the disappearance of the formulation from the site ofadministration. However, it is recognised that at these injection orimplantation sites, as well as at other sites in living tissue, otherdegradation mechanisms may be involved such as those mediated byenzymes.

[0011] Suitable homo- and co-polyesters are those derived fromhydroxy-acids or from the polycondensation of diols and/or polyols, forexample (but not limited to) polyethylene glycols, polypropyleneglycols, 2-10C alkylene glycols, glycerol, trimethylolpropane, andpolyoxyethylated forms of polyfunctional alcohols such as glycerol,trimethylolpropane and sugars, with dicarboxylic acids and/orpolycarboxylic acids, for example (but not limited to) (1-10Calkane)dicarboxylic acids, particularly malonic, succinic and glutaricacids, phthalic acids, mellitic and pyromellitic acids, optionally inthe presence of hydroxy acid(s) and/or mono-ols.

[0012] The preferred methods of preparing homo- and co-polyesters basedupon hydroxy acids are by ring opening polymerisation of the cyclic aciddimers or by direct polycondensation or co-polycondensation of thehydroxy acids or mixtures of the hydroxy acids, or lactones derived fromsuch hydroxy acids. These polymerisations, both of the ring opening typeor the polycondensation type, are preferably carried out so that theresulting homo- or co-polyesters contain, in whole or in part, polymerchains having carboxylic acid functionality. Thus the ring openingpolycondensation of the acid dimers is carried out in the presence of anappropriate polymer chain transfer agent or co-initiator which controlsboth the molecular weight and the structure of the resulting homo- orco-polyester. Suitable such transfer agents are water, hydroxycarboxylicacids, monocarboxylic acids, dicarboxylic acids and polycarboxylicacids.

[0013] For polyesters prepared by polycondensation orco-poly-condensation, the polymerisation is carried out under conditionssuch that an excess of carboxylic acid functionality is used, that is,the ratio of [—COOH] to [—OH] is equal to or greater than 1. Thestructure and molecular weight of the polycondensate are determined bythe nature of the alcohols used (whether mono-ols, diols or polyols, ora mixture), the nature of the acids used (whether mono-, di- orpoly-carboxylic acids, or a mixture), and the amount of the excess ofcarboxylic acid used. Acids involved in the Krebs cycle are particularlyuseful.

[0014] Examples of suitable hydroxy acids or lactones, which may be usedto manufacture homo- or co-polyesters useful in this invention, includeβ-propionolactone, β-butyrolactone, γ-butyrolactone and pivalolactone,and α-hydroxybutyric acid, α-hydroxyisobutyric acid, α-hydroxyvalericacid, α-hydroxyisovaleric acid, α-hydroxycaproic acid,α-hydroxyisocaproic acid, α-hydroxy-β-methylvaleric acid,α-hydroxyheptanoic acid, α-hydroxydecanoic acid, α-hydroxymyristic acidand α-hydroxystearic acid. Preferred such homo- and co-polyesters arethose derived from lactic acid in its D-, L or DL- form, and glycolicacid, or the corresponding dimers lactide and glycolide, and a preferredoptional chain stopper is lactic acid.

[0015] Although a macromolecular, basic peptide drug can exist wholly orin part as a polymer-cation, and a polyester can exist wholly or in partas a polymer-anion, salt formation arising from acid-base interactionbetween such polymeric species, using conventional processes of mixing,or using organic solvents, is extremely difficult or even impossible.For example, melt mixing the two components is unsuitable, since it iswell known that peptides do not normally melt, but rather decompose atthe elevated temperatures commonly used to melt polymers. However, evenif the peptide were to melt (which it does not), it would beincompatible with, or insoluble in, a homo- or co-polyester forthermodynamic reasons, as follows.

[0016] Peptides are macromolecules, and so possess many of the typicalproperties of conventional polymers. They are therefore (in the absenceof specific chemical or physical interactions) totally incompatiblewith, or insoluble in, other macromolecules which have differentchemical and polymer backbone structure, as the free energy of mixing ofthe two dissimilar polymer types is highly positive and so is notthermo-dynamically favoured. In the bulk state, peptides are highlypolar and strongly hydrogen bonded molecules, with the result that theenthalpy of mixing of peptides with homo- or co-polyesters (which arerelatively non-polar, and in which hydrogen bonding is either absent orweak) is highly positive; that is, endothermic and thermodynamically notfavoured. Further, macromolecules are by definition large, and so have alow intrinsic entropy, resulting in the entropy of mixing of twodifferent macromolecular species being very low or even negative. (See,for example, P J Florey, “Principles of Polymer Chemistry”, CornellUniversity, 1953 at 555; L Bohn, “Polymer Handbook”, 2nd Edition, JWiley 1975, III-211; and L Bohn, Rubber Chemistry and Technology, 1966,493). Consequently, the mixing of a peptide with a polyester at elevatedtemperature in the molten state will not give rise to the mixing on themolecular scale necessary for salt formation to occur. Simple admixtureof a peptide and a polyester, therefore, does not give rise to saltformation.

[0017] Similar difficulties exist with attempts to form salts ofpeptides and polyesters using organic solvents, unless the peptide hassome solubility or swellability in the solvent. The solubilityproperties of polyesters and peptides are totally different. Solventswhich dissolve the peptide, such as water, are complete non-solvents forthe polyester; and, in general, good solvents for the polyester, such asdichloromethane, are complete non-solvents for the peptide. Thosesolvents which can dissolve both the peptide and the polyester, such asdimethylsulfoxide, dimethylformamide, dimethylacetamide andN-methylpyrrolidone, have different problems because they are relativelynon-volatile, have high boiling points, and so are extremely difficultto remove, and also because of the unacceptable toxicity of some ofthese solvents. It has been possible to identify certain solvents forboth components which are more volatile and which are toxicologicallyacceptable, but such solvents present other difficulties. For example,acetic acid is a solvent for both peptides and polyesters, but the useof a large amount of acid solvent predisposes the peptide to exist asthe acetate salt (because of mass action effects), so that the removalof the acetic acid at room temperature (say 20-25° C.), or by freezedrying, results in phase separation of the peptide and the polyester, sothat the desired salt formation tends not to occur.

[0018] It is an object of the present invention, therefore, to provide aprocess for the manufacture of a salt, comprising a cation of a basicpeptide and an anion of a carboxy-terminated polyester.

[0019] The preparation of the peptide-polyester salts of this inventioncan be carried out using homo- or co-polyesters containing carboxylicacid groups, and peptides wherein the basic residues occur as the freebase or as salts of a weak acid, preferably a volatile acid, having anacid dissociation constant of less than 10⁻³ or a pK(pK_(a)=−log₁₀K_(a), where K_(a) is the acid dissociation constant) ofgreater than 3. A particularly preferred such basic peptide salt is asalt with acetic acid. However, because of the inherent incompatibilityof the two macromolecular species, particular conditions have to be usedin which these peptide-polyester salts can be generated.

[0020] One means of achieving this is to use a solvent which dissolvesboth the peptide and the polyester, to form a solution, from which thesolvent can be removed directly, leaving either firstly the amphipathicsalt, or secondly a mixture of polyester and peptide in a physical statewhich is predisposed to form the amphipathic salt when processedfurther.

[0021] An example of the first approach is to use solvents such as, butnot limited to, dimethylsulfoxide, dimethylformamide, dimethylacetamideand N-methylpyrrolidone, which are essentially neutral and which can besolvents for both the peptide and the polyester. Under normalcircumstances, as indicated above, these solvents are extremelydifficult to remove, due to their high boiling points and relativenon-volatility. When a peptide (for example as an acetate salt) and apolyester are dissolved in one of these solvents, the peptide tends toexist as the salt with the polyester, as the more strongly acidic lacticor glycolic acid group in the polyester displaces the weaker carboxylicacid. The bulk of the solvent and liberated acetic acid (or other weakbut volatile carboxylic acid) may be removed in vacuo, and the residualsolution containing peptide-polyester salt is added to distilled water,to precipitate the insoluble polymeric salt.

[0022] The distilled water is preferably carbon dioxide-free, to avoidthe formation of carbonate salts by displacement of the polyester anion.Residual solvent in the peptide-polyester salt may then be removed byfurther washing with water, also preferably carbon dioxide-free. In somecircumstances, the polymeric salt may be isolated by directprecipitation into water, without any need to remove any solvent, andthis approach is particularly useful when the peptide is used as thebase.

[0023] Thus, according to a further feature of this invention, there isprovided a process for the manufacture of a salt comprising a basicpeptide and a carboxy-terminated polyester, which comprises dissolvingthe basic peptide, in free base form or in the form of a salt with aweak acid, for example acetic acid, and the carboxy-terminated polyesterin a neutral, polar solvent in which both are soluble, removing thesolvent or most of the solvent, and adding the remaining concentratedsolution to an excess of a non-solvent for the peptide-polyester salt.

[0024] The second approach, also based on using a solvent whichdissolves both the peptide and the polyester, relies on said solventbeing capable of removal by freezing and conventional freeze drying, orby spray drying. An essential part of this process is the removal of thesolvent from the peptide-polyester mixture at an extremely rapid, almostinstantaneous, rate, and preferably at a temperature which is below theglass transition temperature of the polyester and the peptide. In thiscase, the solvent may be neutral or acidic, and a preferred solvent isacetic acid.

[0025] Such extremely rapid removal of solvent from a solution whichexhibits some degree of viscous flow or visco-elastic behaviour resultsin phase separation of the two incompatible macromolecular typesoccurring on an extremely small colloidal scale. That is, the resultingpeptide-polyester mixture has an extremely high surface area and surfaceenergy. As a consequence, when another different solvent for thepolyester, which is normally a non-solvent for the peptide, is added toessentially solvent-free peptide-polyester mixtures of this type, thehigh surface energy is dissipated by salt formation, and thedisappearance of the colloidal nature of the peptide in the polyester.Suitable solvents for this second approach have to be freeze dryable andinclude, but are not limited to, acetic acid, dioxan/water mixtures andtert-butanol/water mixtures, or have to be spray dryable.

[0026] Thus, according to a further feature of this invention, there isprovided a process for the manufacture of a salt comprising a basicpeptide and a carboxy-terminated polyester, which comprises dissolvingthe basic peptide, in free base form or in the form of a salt with aweak acid, for example acetic acid, and the carboxy-terminated polyesterin a solvent in which both are soluble, and which is capable of beingremoved by freeze-drying, freezing the resulting solution at high speed,freeze-drying the resulting frozen mixture, dispersing the resultingmixture in a solvent for the polyester component, and allowing themixture to dissolve as the peptide-polyester salt is formed.

[0027] More particularly, in this process the solution of the peptideand the polylactic acid, or a co-polymer of lactic and glycolic acids,in acetic acid is added to liquid nitrogen in a dropwise fashion. Thisresults in a more or less instantaneous freezing of the acetic acidsolution, and a more or less instantaneous generation of an essentiallysolvent-free peptide-polyester mixture. Freeze-drying to remove theacetic acid solvent gives a peptide-polyester product mixed on anextremely fine colloidal scale. For many peptides, the colloidal natureof such a material is demonstrated when a solvent for the polyester isadded, for example dichloromethane, when an extremely fine colloidalsuspension is generated, and providing there is an excess of carboxylicacid functionality in the mixture, a clear solution can be obtainedeventually on standing, the excess surface energy being lost aspeptide-polyester salt is formed. Other procedures to more or lessinstantly freeze the peptide/polyester/acetic acid mixture may be usedin place of dropwise addition to liquid nitrogen, for example droppingthe mixture into a mixture of solid carbon dioxide and hexane.

[0028] Hypothetically, of course, a totally insoluble compound can bemade to be soluble if it can be reduced to a sufficiently small averageparticle size. If it is assumed that the particle is a sphere of radiusr, having density σ, and that it has a surface energy γ, such a particlewill have a surface energy 4πr²γ associated with it. It will also have amass of {fraction (4/3)}πr³ a and so the surface energy per unit mass is^(3πγ)/_(or).

[0029] Consider now two cases of saturated solutions:

[0030] (i) when excess solid is extremely coarse and therefore has verylittle surface energy and the saturated solution has a concentrationC_(s). Then the Gibbs free energy is:G_(solution)¹ = G₀ + RT  ln   C_(s) = G_(solid)¹;

[0031] (ii) when the excess solid is extremely small particles of radiusr, the Gibbs free energy of the solution which is in equilibrium withextremely small particles is: G_(solution)² = G₀ + RT  ln   C

[0032]  but in this case the solid has a Gibbs free energy of${G_{solid}^{1} + \frac{3\quad \pi \quad \gamma}{\sigma \quad r}},$

$\begin{matrix}{\quad {{{\text{and}\quad G_{solution}^{2}} = {{G_{0} + {{RT}\quad \ln \quad C}} = {G_{solid}^{1} + \frac{3\quad \pi \quad \gamma}{\sigma r}}}},}} \\{\quad {{\text{or}\quad G_{solid}^{1}} = {G_{0} + {{RT}\quad \ln \quad C} - {\frac{3\quad \pi \quad \gamma}{\sigma r}.}}}} \\{\quad \text{But from (i) above,}} \\{\quad {{G_{solid}^{1} = {G_{0} + {{RT}\quad \ln \quad C_{s}}}},}} \\{\quad \text{and~~therefore}} \\{\quad {{{G_{0} + {{RT}\quad \ln \quad C} - \frac{3\quad \pi \quad \gamma}{\sigma r}} = {G_{0} + {{RT}\quad \ln \quad C_{s}}}},}} \\{\quad {{\text{or}\quad C} = {C_{s} \cdot ^{3{{\pi\gamma}/\sigma}\quad r}}}} \\{\quad \text{so~~that,~~as~~r~~decreases,~~C~~(hypothetically)~~increases.}}\end{matrix}$

[0033] In the usual case, higher than normal solubility due to smallparticle size is metastable, and the particles grow in size, for exampleby dissolution and recrystallisation, so that the effect of high surfaceenergy is negated. However, with small particle size peptide-polyestermixtures, salt formation can occur, and this offers an alternative meansof reducing the surface energy of the colloidal particles by allowingthe formation of a soluble amphipathic salt, which as a solution offersthe lowest free energy condition.

[0034] According to a further feature of the invention, there isprovided a process for the manufacture of a salt comprising a basicpeptide and a carboxy-terminated polyester, which comprises reacting abasic peptide in the form of a salt with a strong acid, such as achloride or sulfate, with a polyester wherein some or all of thepolyester is in the form of a carboxylic acid salt with a suitablealkali metal or alkaline earth metal, for example a sodium, potassium,calcium or magnesium carboxylate salt. For low molecular weightpolyesters, (having a weight average molecular weight of less than about10,000), the salts with alkalis can be dissolved, or very finelydispersed, in water. Addition of such a solution or dispersion to anaqueous solution (preferably free of carbon dioxide) of the peptide,results in precipitation of the water-insoluble amphipathicpeptide-polyester salt.

[0035] In a similar way, the chloride or sulfate salts of ‘pegylated’basic peptides (polyoxyethylene conjugates of peptides) are, or can be,partially compatible with, or soluble in, solvents such asdichloromethane, and the sodium or potassium salts of carboxy-terminatedpolyesters can also be soluble in dichloromethane. Thus, when two suchsalts are mixed in the appropriate proportions, the solublepeptide-polyester salt is generated by double decomposition, withprecipitation of the alkali metal chloride or sulfate.

[0036] The thermodynamic incompatibility of different macromolecules,referred to above, has been known for many years, but it has rarelyentered into any consideration in the prior art of the extended releaseof peptide drugs from polyester matrixes. A necessary consequence ofthis thermodynamic incompatibility, or insolubility, is that in normalcircumstances polyesters are totally impermeable to peptide drugs. Forpartition-dependent Fickian diffusion of a peptide drug through apolyester to occur, the peptide must have some solubility in thepolyester. However, for the reasons discussed above, this is not thecase, and so transport of the peptide through the polyester bypartition-dependent Fickian diffusion is impossible.

[0037] Furthermore, even if, for the sake of argument, the peptide drug,or one of its synthetic analogues, had some solubility in orcompatibility with the polyester, transport by diffusion through thepolyester phase would still be impossible. It has long been recognisedthat the free volume in the polyester, which arises from rotational andtranslational polyester segment mobility, and which should allow thepassage of diffusing molecules, is insufficiently large to accommodatethe diffusion of macromolecules having molecular weights greater thanabout 500 Da or so. (See, for example, R W Baker and H K Lonsdale,“Controlled Release: Mechanisms and Rates” in “Controlled Release ofBiologically Active Agents, ed. A C Tanquary and R E Lacey, PlenumPress, 1974, 15 et seq.)

[0038] However, even though transport of a peptide drug through apolyester by Fickian diffusion is essentially impossible for peptides ofmore than about 500 Da or so, continuous release of polypeptides hasnevertheless been achieved. European Patent No. 58,481 discloses howcontinuous release of a peptide drug from a polyester was obtained byusing the very different properties of the two macromolecules, peptidesbeing hydrophilic and water-soluble, and polyesters being hydrophobicand water-insoluble. In the formulations described in that patent,peptide drug release was achieved primarily through aqueous pores, whichare generated initially by simple leaching of peptide from domains atthe surface of the formulation, or from domains of peptide drug whichare continuous or contiguous with the surface of the formulation. Thisleaching provides for an initial phase of release, and subsequent bulkhydrolytic degradation of the polyester results in the generation offurther porosity within the polyester, and so further peptide release,governed by degradation and erosion, can occur. If the porosity arisingfrom hydrolytic polyester degradation does not occur quickly enough, theinitial release from the leaching phase is complete before sufficientdegradation-induced porosity is generated in the delivery system, anddiscontinuous release of the peptide is obtained. The parameters of theformulations disclosed in EP 58,481 were therefore chosen so thathydrolytic degradation of the polyester occurred at the right time inrelation to the initial leaching release phase, so as to ensure that thetwo phases of release overlapped, resulting in continuous release of thepeptide drug.

[0039] However, whereas Fickian diffusional transport of a peptidethrough the polyester phase is impossible in the case of those simplepeptide-polyester mixtures, a totally different situation arises in thecase of formulations of the peptide-polyester salts of the presentinvention, optionally in the presence of free polymer. In formulationscontaining these materials, there is no separate phase consisting ofpolyester alone; rather, the continuous phase which controls release ofthe peptide is wholly or in part the peptide-polyester salt. Freepeptide has some solubility in this phase of peptide-polyester salt, andso in formulations using such materials, true Fickian,partition-dependent diffusion of a peptide is possible, if the otherrequirements, such as effective free volume, are present.

[0040] Because the peptide-polyester salt contains a highly hydrophilicsegment, the peptide-polyester salt formulation has a much higher wateruptake than the polyester alone. Furthermore, in these formulations thewater uptake is enhanced even more, due to the ionic nature of thepeptide-polyester interaction, and the solvation of ions or ion pairs inthe macromolecular salt by water. This implies an essentially hydrogelnature for the peptide-polyester salt, and provides an increase in thedegrees of mobility of macromolecular segments in thepolycation-polyanion complex. That is, the effective free volume of thematrix material is increased, and so can accommodate a macromolecularpeptide.

[0041] The net effect of these properties of the peptide-polyester salt,(optionally in the presence of free polymer), is to allow Fickiandiffusional transport of a macromolecular peptide through the matrix ofpeptide-polyester salt or the mixed salt and free polymer phase. This isa totally different situation from that which occurs with polyesteralone, or with simple admixtures of peptides and polyesters, and soextended release matrixes or membranes based on the increasedpermeability arising from the use of the peptide-polyester salt arecentral to the formulations for the controlled release of peptidesdescribed hereafter in this application.

[0042] The peptide-polyester salts of the present invention thus providenew and unexpected advantages in the design of parenteral drug deliverysystems, based on solutions or dispersions using various mixtures offree peptide drugs, free polyester and peptide-polyester salt, in bothaqueous and non-aqueous pharmaceutically acceptable injection vehicles,and based on sub-dermal implants which can be injected, intramuscularlyor sub-cutaneously, or implanted, by virtue of the novel and unexpectedsolubility of these peptide-containing moieties in lipophilic solvents.Furthermore, formulations based on these peptide-polyester salts, inparticular those using highly lipophilic polyesters, can also beadministered by other routes. Of particular importance is the oralroute, in which the various combinations of peptide-polyester saltand/or free peptide drug and/or free polyester can be used to goodeffect. In many instances, for oral administration it is preferred touse a pharmaceutically acceptable carrier such as a vegetable oil or avariant thereof, and including mono-, di- and tri-glycerides eitheralone or in admixture with other oils. Of less importance are thetopical, rectal and intranasal routes of administration.

[0043] Other than European Patent No. 58,481 (1982), referred to above,Lawter et al. (loc. cit.) and Okada et al. (loc. cit.) are the onlystate of the art known to the applicants herein which refers to thepossibility of obtaining peptide-polyester salts, but both thesepublications are speculative, in that they do not disclose how thisputative interaction can be realised or utilized. It is a further objectof the present invention to provide extended release pharmaceuticalformulations, comprising various combinations of peptide-polyester saltand/or free peptide drug and/or free polyester in various proportions togive at least three different profiles of controlled drug release.

[0044] Thus, according to a further feature of the invention there isprovided an extended release pharmaceutical composition comprising apeptide-polyester salt, as defined above, and/or free peptide drugand/or free polyester, and optionally other pharmaceutical excipient orexcipients.

[0045] The design of the pharmaceutical compositions of this inventionis based upon the following considerations. Whereas a simple peptidedrug is normally soluble in water, both its salt with a polyester, andthe free polyester itself, are normally totally water-insoluble,(although it is recognised that, for very low oligomeric forms ofpolyesters and co-polyesters, whilst they may themselves bewater-insoluble, they may be water-soluble when in the form of apeptide-polyester salt). However, incubation of a mixture of a peptidedrug and a polyester, wherein all or part of the peptide is present asthe peptide-polyester salt, in aqueous physiological fluids, results insome degradation of the polyester. If these degraded products arewater-insoluble, then the degrading peptide-polyester salt will continueto be insoluble. On the other hand, if the polyester is of sufficientlylow molecular weight initially, or contains a polymeric component ofequally or similarly low molecular weight, such that water-solublepolyester-derived acidic fragments are produced, then these fragments(as anions) are co-transportable with the polypeptide cation. It hasbeen shown for the new peptide-polyester salt compositions of thisinvention that immediacy of release is strongly dependent on themolecular weight and molecular weight distribution of the polyestercomponent.

[0046] Molecular weight distribution is defined as $\begin{matrix}{\quad \frac{M_{w}}{M_{n}}} \\{{{\text{where}\quad M_{w}\quad \text{(weight~~average~~molecular~~weight)}} = {\frac{\Sigma \quad {w_{i} \cdot M_{i}}}{\Sigma \quad w_{i}} = \frac{\Sigma \quad {n_{i} \cdot M_{i}^{2}}}{\Sigma \quad {n_{i} \cdot M_{i}}}}}\quad} \\{{\text{and}\quad M_{n}\quad \text{(number~~average~~molecular~~weight)}} = \frac{\Sigma \quad {n_{i} \cdot M_{i}}}{\Sigma \quad n_{i}}}\end{matrix}$

[0047] and where w_(i) is the weight fraction of polymer moleculeshaving a molecular weight M_(i), and n_(i) is the number of polymermolecules having molecular weight M_(i).

[0048] Molecular weight distribution is often referred to aspoly-dispersity, and the various values for narrow, normal or mostprobable, and wide distribution are well known (see, for example,“Polymer Handbook”, 2nd Edition, J Wiley 1975, IV-3.) It is generallyaccepted that a polydispersity of less than 1.8 is a narrow distributionor low polydispersity, approximately 1.8 to 2.2 is a normal or mostprobable distribution or normal polydispersity, and more thanapproximately 2.2 is a wide or broad distribution or highpolydispersity.

[0049] For the administration of peptide drugs by the parenteral route,such as intramuscular or sub-cutaneous injection or sub-dermalimplantation of a depot or delivery system, polyesters having a numberaverage molecular weight of more than 2000 Da, or an inherent viscosityat 1%w/v at 25° C. in chloroform of more than or equal to 0.08 dl/g, andup to and including 4.0dl/g, are preferred. For administration by otherroutes, such as orally, the preferred range of number average molecularweight is 500 to 5000 Da.

[0050] It is obvious from the above considerations, which have largelybeen ignored in the state of the art, that the degradation of thepolyesters, particularly in the presence of basic peptide, to give evena small fraction of water-soluble derived fragments, and the timeinterval for this to occur, will be controlled by molecular weight andmolecular weight distribution. Essentially immediate degradation towater-soluble fragments occurs using both narrow and normal distributionpolyesters, having weight average molecular weights of less than about10,000 Da and less than about 15,000 Da respectively (depending on thetype of molecular weight distribution), but in general the lower thepolydispersity of the polyester the lower the weight average molecularweight required for immediate degradation to water-soluble fragments.For polyesters of weight average molecular weight of greater than 15,000Da, normal or wide distributions are required. Again this depends inpart on the nature and type of the molecular weight distribution, but ingeneral the higher the weight average molecular weight, the higher thepolydispersity needed in order to achieve early degradation towater-soluble fragments.

[0051] For polyester or co-polyester and peptide compositions where someor all the peptide is in the form of a peptide-polyester salt,optionally containing free polyester, three different release profilescan be obtained. The first of these is when degradation of the polyesteroccurs to give essentially immediate generation of acidic water-solubleor hydrophilic fragments, which results in immediate release of peptideaccording to the following mechanism:

[0052] In this first case, the composition either may contain all thedrug as peptide-polyester salt, or it may contain some free, unbounddrug in addition to some peptide-polyester salt, in both cases alsooptionally in the presence of free polymer. However, the polymerdegrades to water-soluble fragments, in the presence of peptide, almostimmediately, with the consequence that almost immediate sustainedcontinuous release of the peptide commences. It is to be noted that thediffusion of the free water-soluble peptide through the degradingcomposition is facilitated by the increased permeability of the matrixdue to the presence of the peptide-polyester salt in the continuousphase that modulates release.

[0053] The second of these cases is when all the peptide drug is presentas the peptide-polyester salt (optionally in the presence of freepolyester), but the polyester does not degrade immediately towater-soluble fragments. This results in an initial interval in whichthere is no release of peptide drug. Even though the peptide-polyestersalt confers on the matrix increased permeability to free diffusingpeptide, there is no free peptide drug available to diffuse. All thepeptide is in the form of a water-insoluble peptide-polyester salt, andit is only after some considerable time that the polyester degrades towater-soluble fragments and gives rise to free and transportable drug.This results in an extended induction period, during which there isinitially no peptide release, following which induction period, releasecommences. This second case is ideal for timed and pulsed release ofsoluble vaccines and peptides.

[0054] The third case is when a formulation, based on apeptide-polyester drug system which contains a peptide drug both in itsfree form and in the form of a polymer-drug salt, optionally also in thepresence of free polyester, and in which the polyester has a weightaverage molecular weight of greater than about 15,000Da, (and preferablygreater than about 30,000 Da), and having a narrow, or most probable,molecular weight distribution, is placed in a physiological environment,such as is found at intramuscular and sub-cutaneous injection sites,discontinuous release can result. A first phase of release arisesbecause of the presence of free peptide drug, and its ability to betransported through the more permeable peptide-polyester salt system. Ifthis first phase of release of free peptide drug is complete beforedegradation of the polyester in the peptide-polyester salt occurs togive further free peptide drug, then discontinuous peptide drug releasewill ensue.

[0055] Obviously, if there is no interval in which free peptide drug isabsent from the composition, during its degradation, then continuousrelease will be obtained. This release profile is similar to thatdisclosed in European Patent No. 58,481, but the mechanism of release inEuropean Patent No. 58,481 and the materials used (no peptide-polyestersalt) are quite different from the mechanisms and materials defined inthis application. Depending on release profile these mixtures are idealfor continuous release of peptides, proteins and soluble vaccines.

[0056] As stated above, these peptide-polyester drug salt systems, theirphysicochemical characteristics and the mechanisms by which release ofthe peptide occurs, are quite different from those disclosed in EuropeanPatents Nos. 58,481 and 52,510, and all other publications relating topeptide release from homo- and co-polymers of lactic and glycolic acids,which are known to the inventor hereof. Of these only European PatentNo. 58,481, Lawter et al (loc. cit.) and Okada et al (loc. cit.) makeany reference to salt formation arising from the ionic interaction ofpolyester carboxylic acids groups and basic amino acids in peptides, butthe composition made as described therein contain no peptidedrug/polyester salt. These prior disclosures, however, are speculativein this regard, and do not establish conclusively that such interactionsdo indeed occur, nor do they demonstrate how such peptide-polyestersalts can be prepared and isolated, and then used to effect the releaseof peptides, with a variety of different profiles of release, by virtueof their unexpected solubility in lipophilic organic solvents.

[0057] Amongst the properties of peptide-polyester mixtures that willdetermine release, and which have not been mentioned hitherto, are thenumber of basic functional groups in the peptide and the number ofcarboxylic acid groups in the polyester. The above-mentionedpublications are also silent with regard to the remarkable andunexpected effects arising from the use of the peptide-polyester salts,and the surprisingly high permeability of systems containing, in wholeor in part, the peptide-polyester salt, compared with the permeabilityof the polyester alone, or mixtures in which the two components aresimply mixed, and which therefore contain no peptide-polyester salt.

[0058] This difference in permeability can be demonstrated in simplediffusion cell experiments, wherein a continuous and fault-freepolyester membrane, separating two aqueous compartments, one containingan aqueous peptide solution and the other containing the aqueous phasealone, will not allow peptide transport across it, prior to significantdegradation of the membrane polyester. In contrast, membranescontaining, wholly or in part, the peptide-polyester salt allows drugtransport across the salt-containing membrane by partition dependentdiffusion, even if the peptide has a molecular weight of greater than500 Da.

[0059] The peptide-polyester salts of the invention have many othersurprising and useful advantageous properties, unknown in any similarprior art materials, which are particularly useful in the design andmanufacture of pharmaceutical delivery systems. One of the most usefulof these properties is the good solubility of the peptide, when in theform of a polyester salt, in organic solvents in which peptides arenormally totally insoluble. This offers a great many advantages inpharmaceutical manufacture, in that it allows new processes andprocedures to be used for the manufacture of drug delivery systems, andparticularly facilitates aseptic manufacture. These processes andprocedures, and the materials used, are totally different from theprocedures and materials disclosed in the prior art.

[0060] Thus, solutions of a peptide-polyester salt, optionallycontaining free polymer, and/or free peptide in a solubilised ordispersed form, can be sterile-filtered, thus easing the problemsnormally associated with the sterile manufacture of solid or suspensionpeptide formulations. A sterile-filtered solution of a peptide-polyestersalt can therefore be subjected to a variety of pharmaceutical dryingprocedures in an aseptic environment. Spray-drying, spray-congealing andother drying procedures which generate solid particles are preferredprocesses which readily lend themselves to aseptic operation.

[0061] Particularly useful is the generation of microparticles havingparticle sizes in the range from 0.2 μm to 500 μm, which can besuspended in a pharmaceutically acceptable injection vehicle. Suchmicroparticles can be suspended in an aqueous injection vehicle prior touse, or alternatively in an organic injection vehicle which is anon-solvent for the materials used. For delivery systems based on homo-and co-polymers of lactic and glycolic acids, suitable such organicvehicles are highly lipophilic oils, such as (but not limited to) ethyloleate, isopropyl myristate, vegetable oils and various fattyglycerides. In certain circumstances, it is preferred to use mixtures ofsuch lipophilic vehicles.

[0062] Although such lipophilic vehicles are non-solvents for deliveryforms based on lactic and glycolic acids, they are unsuitable for usewith highly lipophilic polyesters such as those based on long chainhydroxy acids, for example hydroxystearic acids. For such highlylipophilic polyesters or co-polyesters, hydrophilic organic injectionvehicles are preferred, such as (but not limited to) propylene glycoland low molecular weight polyethylene glycol. Obviously, aqueousinjection vehicles are also suitable for delivery systems based on themore lipophilic polymers.

[0063] An alternative means of making microparticles utilises anotherunexpected and advantageous property of the peptide-polyester salts ofthis invention. The peptide-polyester salt is comprised of a hydrophilicpeptide, which would prefer thermodynamically to exist or dissolve in anaqueous or polar environment or phase, and a polyester chain which ishydrophobic, and would prefer thermodynamically to dissolve in ahydrophobic phase. That is, the peptide-polyester salt is amphipathic,and has surface-active properties which are not present in simplepeptide salts. This surface activity results in the peptide-polyestersalt preferring to exist at a phase interface, and because of thegeneral nature of the salt (proportion and length of the hydrophobicchain) the most thermodynamically stable type of dispersion in a largelyaqueous phase is for the peptide-polyester salt to exist as a dispersionin water (as the critical micellar concentration is very low, and notall the salt can exist at the interface in many situations.)

[0064] It can be seen, therefore, that the peptide-polyester salt is anextremely effective dispersant for making, as well as for maintaining,the stability of aqueous dispersions. In this second procedure formaking microparticulate pharmaceutical formulations, thepeptide-polyester solution (say, for example, in dichloromethane) issimply dispersed in an aqueous phase, which may optionally contain aviscosity-enhancing polymer such as (but not limited to) polyvinylalcohol, using the surface-active properties of the peptide-polyestersalt. Although some organic solutions containing such peptide-polyestersalts may spontaneously disperse, as a general rule some agitation orshear is required in preparing the aqueous dispersion.

[0065] A further preferred aspect of the process, as indicated above, isto carry out the operations such that the aqueous dispersion is carriedout effectively in the absence of carbon dioxide and in an inertatmosphere. It is further preferred that the organic solution of thepeptide-polyester salt be free of carbon dioxide, because theconcentration of carbon dioxide in air and water under normal conditionsis sufficiently high, in comparison with the concentrations of polyestercarboxylic acid groups, to enter into competitive salt formation due tomass action effects, according to the equation:

[0066] where P is polyester and D is peptide drug. The resultant aqueousdispersions may then be dried by a variety of techniques, such asremoval of the organic solvent in vacuo followed by freeze drying, or bydirectly removing both the solvent and the water in a single freezedrying operation. The resultant product may then be used to makesuitable pharmaceutical preparations for injection in the mannerdescribed above.

[0067] A further alternative means of making microparticulatepharmaceutical formulations uses an essentially dry solution of thepeptide-polyester salt, containing colloidally dispersed free peptide,in a suitable organic solvent or vehicle. (The term “essentially dry” isused, as it is virtually impossible to remove all traces of water fromthe peptide, and furthermore it means that none of the drug exists as anaqueous solution in a separate aqueous phase.) Addition of a non-solventfor the polymer, under conditions of vigorous agitation, followed by theaddition of the solvent-swollen peptide-polyester salt (optionallycontaining free polymer and optionally containing free drug) to a largevolume of a second non-solvent, to further harden and stabilise theprecipitated microparticles, gives the final form. Obviously, under theappropriate conditions, or in the presence of a suitable surface activeagents, such as (but not limited to) the fatty acid esters of sorbitol,the precipitation of the microparticles can be carried out using asingle non-solvent for the polyester, for example a paraffin such ashexane.

[0068] The microparticles made by the various processes described hereinare totally different structurally from the microcapsules preparedaccording to the methods outlined in European Patent Nos. 52,510(Syntex) and 145,240 (Takeda), wherein the peptides are encapsulated ina phase of polyester alone. Hicrocapsules are defined as one or morecores of one compound or material within a continuous second phase, sothat a continuous coating of the second phase material totally enclosesor microencapsulates the core material such that none of that materialexists at the surface of the microcapsules, and microencapsulated corematerial retains in all respects the physicochemical and thermodynamicproperties of the unencapsulated core compound or material.

[0069] Thus, in European Patent No. 52,510, a phase separationcoacervation process was used to coat droplets of an aqueous dilutesolution of the peptide such that the polymer alone comprised acontinuous coating around the aqueous droplets. That is, they are truemicrocapsules which have the geometry and shape of microspheres. Afterisolating the precipitated microcapsules and hardening and drying, aproduct was obtained wherein the peptide drug exists as a discrete coreor cores within a polymer envelope. Because of the presence of water inthe interior of the microcapsule prior to drying, its removal during thedehydration process at a temperature which is below the glass transitiontemperature of the polymer can result in a particle which is highlyforaminous. At no stage does the process and materials, used ordescribed in European Patent No. 52,510, involve a peptide-polyestersalt, nor does the disclosed process allow of sterile filtration of apeptide-polyester solution or suspension, if aseptic manufacture isrequired.

[0070] Furthermore, this prior patent specifically used the polyestersbased on lactic and/or glycolic acids described in U.S. Pat. No.3,773,919 (Boswell), which are defined therein as being benzene-solubleat 25° C. In the present invention, benzene-insoluble polyesters, basedon lactic and/or glycolic acids, but which are soluble in chloroform,are preferred for relatively short delivery periods, say less than twomonths.

[0071] In European Patent No. 190,833 (Takeda), the peptide wasentrapped as a gelled aqueous solution of drug, and the aqueous gelledphase was dispersed in a polymer solution. This water(aqueous druggel)-in-oil (polymer solution) dispersion was then itself dispersedunder shear in water, to give a water-in-oil-in-water double dispersion.After removal of the organic solvent under vacuum, and lyophilisation,microcapsules were obtained wherein the drug/gelling agent wasencapsulated by polymer alone. The products of this process retain thedrug as the simple salt, and not as the polymer salt of the peptide. Thepharmaceutical formulations of the present invention therefore havestructures, physicochemical characteristics and thermodynamicproperties, which are totally different from the products described inEuropean Patents Nos. 52,510, 145,240 and 190,833, wherein themicrocapsules have the shape and geometry of microspheres in which acore, or cores, of drug is totally enclosed by polymer alone.

[0072] The products of this present application can also have thegeometry and shape of (but are not limited to) microspheres, but eitherthey are not microcapsules at all as defined above but rather aresolutions of peptide-polyester salt (optionally also containing freepolymer), or they are microcapsules wherein free peptide drug isencapsulated within a continuous phase or coating of the polymer-drugsalt, optionally also containing free polymer. As indicated above, thepermeability properties of such a polymer-drug salt are totallydifferent from those of free polymer alone, so the products of thepresent invention release their peptide drug load in a manner which istotally different from those described in prior European Patents Nos.52,510, 145,240 and 190,833.

[0073] Thus, a further embodiment of the invention is the preparation ofeither microspheres which are not microcapsules, using a solution of thepeptide-polyester salt, optionally containing free polymer, or thepreparation of microspheres which are microcapsules, but which comprisefree drug encapsulated by a phase or coating of peptide-polyester salt,optionally containing free polymer.

[0074] Such diverse particles can be made by a variety of differentprocesses such as precipitation, phase separation coacervation, spraydrying and spray congealing. The preferred particle size ranges from 0.2μm to 500 μm, and said particles can be injected as a suspension in asuitable injection vehicle.

[0075] Particularly effective and useful parenteral pharmaceuticalformulations of peptide drugs can also be prepared in the form ofsolutions of a drug-polyester salt, optionally containing free polyesterand optionally containing dispersed or solubilised free drug, in apharmaceutically acceptable organic solvent which is a solvent for thefree polyester but a non-solvent for peptides and simple salts thereof,such as for example chlorides and acetates.

[0076] Thus, according to the present invention, however, there isprovided a pharmaceutical composition comprising a peptide drug and apolyester, for extended release of the peptide drug, characterized inthat the composition is in the form of a solution, comprising:

[0077] (a) a basic peptide drug, as hereinbefore defined, having amolecular weight of at least 300 Da, and preferably at least 800 Da,which is in the form of a salt with the polyester, the salt comprising acation of the basic peptide and an anion of a carboxy-terminatedpolyester,

[0078] (b) a pharmaceutically acceptable organic solvent which is asolvent for the free polyester but not a solvent for the free peptide,

[0079] (c) an excess of the polyester, and optionally

[0080] (d) an excess of the free peptide drug in a solubilised orcolloidally dispersed form.

[0081] Suitable basic peptides and carboxy-terminated polyesters arethose defined above, and particularly preferred peptides are thosesynthetic LHRH analogues defined above.

[0082] For polyester-peptide drug salts wherein the polyester is basedon homo- and co-polymers of lactic and glycolic acids, suitablepharmaceutically acceptable organic solvents include, but are notlimited to, benzyl benzoate, benzyl alcohol, ethyl lactate, glyceryltriacetate, esters of citric acid, and low molecular weight (<1000)polyethylene glycols, alkoxypolyethylene glycols and polyethylene glycolacetates, etc., and of these benzyl benzoate and benzyl alcohol arepreferred, especially benzyl benzoate.

[0083] The only requirement for such an organic solvent is that it ispharmaceutically acceptable and that the polyester-peptide drug salt issoluble in it. Whether or not a single such solvent is used, or amixture of such solvents, the suitability of such solvents can bedetermined readily by simple experimentation. Homo- and co-polymers oflactic and glycolic acid are amongst the most polar and lipophobicpolyesters, and so will not dissolve in such organic injection solventsas ethyl oleate, vegetable oils and other lipophilic carriers, but homo-and co-polymers based on lipophilic monomers or co-monomers, orlipophilic hydroxy acids such as hydroxystearic acid, are soluble insuch lipophilic injection vehicles.

[0084] The ratio of peptide drug to polyester in the solids which aredissolved to form the solution composition of the invention, willnaturally vary according to the potency of the peptide drug, the natureof the polyester used, and the period of peptide drug release desired.

[0085] The preferred level of peptide drug incorporation is from 0.1 to30%w/v. In general, the optimal drug loading is dependent upon themolecular weight of the polyester and its molecular weight distribution,the period of release desired, and the potency of the peptide drug.Obviously, for drugs of relatively low potency, higher levels ofincorporation may be required.

[0086] Water uptake by the composition is an important factor incontrolling the rate of hydrolytic scission of the polyester, and therate of water uptake is to some degree determined by the drug loading onthe composition. Thus, in cases where relatively rapid drug release isrequired over a relatively short period, say three months, up to 30%peptide drug loading may be appropriate.

[0087] The monomer composition of a co-polyester, for example the ratioof lactide to glycolide in lactide-co-glycolide polyesters, is alsoimportant in determining the rates of polyester degradation and peptidedrug release. Duration of release is also determined in part by theweight average molecular weight of the polyester, but the amount ofpeptide drug which can be incorporated as drug-polyester salt isdetermined by the number average molecular weight. That is,polydispersity (the ratio of weight average to number average molecularweights) is an important parameter.

[0088] Thus, for durations of peptide drug release of from one to fourmonths, compositions comprising polyesters of weight average molecularweight from 4000 to 20000 with polydispersities of from 1.2 to 2.2, andpeptide drug contents of from 0.1 to 30% are preferred. In general, thelower the drug loading, the lower the weight average molecular weightand the higher the polydispersity of the polyester are required. Forlonger release periods, say from two to six months, it is preferred touse peptide drug loadings of from 0.1 to 20%, and polyesters havingweight average molecular weights of 8000 to 20000, and polydispersitiesof from 1.5 to >2.2. For release periods of greater than six months,peptide drug loadings of from 0.1 to 10% are preferred, suing polyestershaving a weight average molecular weight of from 20000 to 50000, andpolydispersities of >1.8.

[0089] The level of incorporation of total peptide-polyester solids inthe composition of the invention will naturally vary, depending upon thepotency of the peptide component, the period of time over which deliveryof the peptide drug is desired, the solubility of the total solids inthe solvent of choice, and the volume and viscosity of the solutioncomposition which it is desired to administer.

[0090] The viscosity of the solution composition of the invention isdetermined by the molecular weight of the polyester and the peptide drugloading. In general, solutions containing over about 40% solids w/v(peptide drug/polyester salt, free drug, free polyester) and where thepolyester has a weight average molecular weight of >8000, are difficultto administer by injection because of their viscosity. Thus solutions of<40% w/v are preferred for these polyesters. For solution compositionscomprising polyesters of weight average molecular weight from about 8000to about 20000, concentrations of ≦0 w/v are preferred, and for solutioncompositions comprising polyesters of molecular weight from about 20000to about 50000, concentrations of ≦0% w/v are preferred. In somecircumstances, for example if it is desired to inject the compositionusing a very narrow needle, very low viscosity solutions may bepreferred, and the concentration could be reduced to 2% w/v or evenless, but there will be a balance, of course, between reducing theviscosity and increasing the volume required to be injected.

[0091] According to a further feature of the invention, there isprovided a process for the manufacture of a composition of theinvention, which comprises:

[0092] 1. dissolving an intimate mixture of the basic peptide drug andthe polyester in the pharmaceutically acceptable solvent; or

[0093] 2. slowly adding a solution of the peptide drug in a 1-6C alkanolto a solution of the polyester in a solvent suitable for injection,whereafter if the hydroxylic solvent is not pharmaceutically acceptablefor injection it is removed by evaporation, or if the hydroxylic solventis pharmaceutically acceptable for injection, its removal may not benecessary.

[0094] The intimate mixture of the basic peptide drug and the polyester,used in process 1. above, is preferably obtained by dissolving the basicpeptide and the polyester in a solvent or solvent mixture which iscapable of dissolving both the basic peptide drug and the polyester, andwhich is capable of being freeze-dried. Suitable examples of suchsolvents or solvent mixtures are glacial acetic acid and mixtures ofdioxan and water, followed by freeze drying of the solution so obtained.Alternatively, the two components may be dissolved in for exampledimethylsulfoxide, and the solvent subsequently removed.

[0095] The intimate mixture may also be obtained by dissolving thepeptide drug in a hydroxylic solvent, for example methanol, and addingthis solution to a solution of the polyester in for exampledichloromethane, followed by removal of the solvents, for example byevaporation.

[0096] Alternatively, an aqueous solution of the peptide drug as thechloride salt may be added to an aqueous solution or dispersion of thesodium salt of the polyester, and the mixture freeze dried to give amixture of the peptide drug/polyester salt and sodium chloride. Thelatter may be removed if desried by mixing the product in an organicsolvent and filtering off the insoluble sodium chloride.

[0097] In process 1., dissolution of the intimate mixture in thepharmaceutically acceptable solvent may be hastened by heating and/orstirring of the reaction mixture.

[0098] In process 2. above, a suitable alkanol solvent for the peptideis for example, methanol, ethanol or propylene-1,2-diol.

[0099] A major advantage of pharmaceutical peptide drug products in theform of solutions of a polyester-peptide drug salt, optionallycontaining free drug and/or free polyester, is that preparation of aninjectable product in sterile form, for immediate use without any needfor premixing prior to administration to a patient, can be manufacturedusing sterile filtration. This is a much simpler manufacturing operationthan the sterilisation of a solid or suspension product. An alternativeprocess for the manufacture of sterile injectable solutions is todissolve a sterile polyester-peptide drug salt, optionally containingfree drug and/or free polyester, in the pharmaceutically acceptableorganic injection vehicle.

[0100] Although these formulations are primarily those for parenteralroutes of administration, the polyester-drug salts of the invention mayalso be used in the manufacture of orally administrable formulations.

[0101] A quite different type of formulation, which can be injected orimplanted sub-dermally, is a drug delivery system based on implants ormixtures of different types of implant. These can be prepared from thepolyester-peptide drug salts of the invention, optionally containingfree drug and/or free polyester, using conventional polymermelt-processing techniques, such as, but not limited to, extrusion, andcompression and injection moulding, wherein elevated temperatures(preferably less than 100° C.) are used to melt the polyester-drug saltin the preparation of the implant. Preparations of such implants can becarried out under aseptic conditions, or alternatively by terminalsterilisation by irradiation, using but not limited to γ- or X-rays.These solid dosage forms can be reduced to microparticulate forms bycomminution or milling. The preferred particle sizes may range from 1 μmto 500 μm, and these microparticle delivery systems (which are neithermicrospheres nor microcapsules) can be suspended in a suitableconventional pharmaceutically acceptable injection vehicle.

[0102] The melt-processing of the peptide-polyester drug salt embodiesand illustrates a most significant and important difference between thephysicochemical and thermodynamic properties of the peptide-polyesterdrug salts of this invention, and the free peptides and simple saltsthereof. The peptide-polyester salts of this invention in many instancesmelt and flow, in contrast to the free peptides and their simple salts,such as chlorides and acetates, which do not melt, but decompose atelevated temperature.

[0103] Degradation of polyesters is in part dependent on their molecularweight and polydispersity. Obviously, for degradation to occur mainly byhydrolytic scission of ester groups, the polyester or a pharmaceuticalformulation containing a polyester, must take up water. For thosesystems where the release controlling matrix or membrane contains, inwhole or in part, peptide-polyester drug salt, there will be a higherwater uptake by the controlling matrix or membrane when compared to thepolyester alone. Consequently, continuous matrix phases or membranescontaining polyester-drug salt degrade differently from those continuousmatrix phases or membranes based on polyester alone. It will also beunderstood that the rate of diffusion of water or physiological fluidsinto such a release controlling polyester matrix or membrane willcontrol in part the rate of degradation. This diffusion of water orphysiological fluids is also governed by the dimensions and shape of theformulation, and so drug release from compositions containing polymericsalts of polypeptides and polyesters is also dependent on these factors.

[0104] Of particular interest as the polyester component of thepeptide-polyester drug salts of this invention, are those based on homo-and co-polymers of lactic and glycolic acids, wherein the lactic may bein any one or more of its optically active and racemic forms. Polyestersof this general type have been known for many years and have beenstudied in detail in a variety of controlled release drug deliverysystems (see, for example, “Controlled Release of Bioactive Agents fromLactide/Glycolide Polymers”, by D H Lewis in “Biodegradable Polymers asDrug Delivery Systems”, ed. M Chasin & R Langer, Marcel Dekker, andreferences therein).

[0105] For example, U.S. Pat. No. 3,773,919 indicates in broad generalterms that controlled release pharmaceutical formulations of lactidepolyesters and lactide co-polyesters containing antimicrobialpolypeptides might be prepared. However, the antimicrobial peptidesdisclosed therein are unsatisfactory for generating a polyester salt,since they either occur as sulfates, or have other features whichinhibit or prevent the formation of a polyester-peptide drug salt.Indeed, when the Examples shown in this patent are followed, the mixingof the peptide drug, irrespective of its nature, with a polymer at anelevated temperature as disclosed, results in catastrophic decompositionof the peptide drug.

[0106] Similarly, an antimicrobial polypeptide, colistin, is disclosedin European Patent No. 25,698 as one of many listed compounds whichallegedly may be formulated with polylactide, but once again thiscompound has structural features which prevent salt formation with theterminal carboxylic acid groups of the polyester. Colistin is usedpharmaceutically only as colistin sulfate or colistin sulfomethatesodium, neither of which forms allows the manufacture of amphipathicsalts with polyesters according to the present invention. Other priorart which discloses the use of polypeptides with biodegradable polymersbased on homo- and co-polymers of lactic and glycolic acids are EuropeanPatents Nos. 52,510, 58,481, 145,240 and 190,833, previously referred toabove.

[0107] Although co-polymers of lactic and glycolic acids have been knownfor many years, the complexity of their structure with regard to thedistribution of the co-monomer units and their subsequent sequencelength (runs of the same individual co-monomer unit in the co-polymer,which are other than random), and the effect of such structuralvariations when used as drug release matrixes, have largely been ignoredin the prior art. This co-polymer structure determines, in part, boththe solubility or swellability of the polymer in solvents such asbenzene, as well as the rate of degradation. This correlation was firstnoted by Hutchinson (European Patent No. 58,481), but has been extendedand refined in the present invention.

[0108] To illustrate this point, U.S. Pat. No. 3,773,919 disclosescertain controlled release drug formulations using 50/50 co-polyestersof lactic and glycolic acids which are soluble in benzene, and indeedthis U.S. patent is specifically limited (in respect of lactic/glycoliccopolymers) to those which are benzene-soluble. The utility of thesebenzene-soluble co-polyesters has been further reinforced by theirspecific use in European Patent No. 52,510. However, earlier U.S. Pat.No. 2,703,316, (which was commonly owned with U.S. Pat. No. 3,773,919)disclosed 50/50 lactide/glycolide co-polyesters which were insoluble inbenzene. Since these two U.S. patents were commonly owned (duPont), itmust be assumed that, in the invention claimed in the later of thesepatents, the benzene-insoluble co-polymers were inferior in some respectas compared to those which were benzene-soluble. This view is reinforcedby European Patent No. 52,510, which used only the benzene-solubleco-polymers of U.S. Pat. No. 3,773,919.

[0109] The prior art, with the exception of our own European Patent No.58,481, has ignored the effect which the structure of co-polyesters oflactic and glycolic acids has on their solubility and degradability. Wehave shown that for polyesters of similar molecular weight and molecularweight distribution the following general relationship applies in mostcases for polyesters which are soluble in chloroform at 25° C., namelybenzene-insoluble polyesters degrade faster than polyesters which areswollen but not dissolved by benzene, and such benzene-swellablepolyesters degrade faster than those polyesters which are freely solublein benzene, when degradation experiments are carried out in aqueousphysiological fluids, or in buffer at pH 7.4 at 37° C. Consequently, itis particularly useful to use polyesters which are insoluble in benzeneto provide continuous release of peptides from parenteral formulationsover a relatively short period of time, say from one week to two months.

[0110] Thus, for compositions which may contain from 0.1% w/v of peptideup to 75% w/v of peptide, the following holds with respect to polyestercomposition, and its relations to structure, viscosity andpolydispersity.

[0111] For the manufacture of peptide-polyester drug salts which can beformulated in accordance with this invention to give continuous drugrelease over a period of a week to two months, the molar composition ofsuch benzene-insoluble polyesters, which preferably have a normal towide polydispersity, preferably ranges from 60% glycolic acid (orglycolide)/40% lactic acid (or lactide) to about 25% glycolic acid (orglycolide)/75% lactic acid (or lactide), and such polyesters preferablyhave an inherent viscosity at 1% w/v in chloroform at 25° C. rangingfrom 0.08 to 4.0 dl/g.

[0112] By suitable choice of the polyester parameters, includingmolecular weight and molecular weight distribution, it is also possibleto achieve continuous release of polypeptides over a period of one weekto two months from formulations according to this invention, usingpolylactic acid homopolymer or co-polyesters having a molar compositionranging from 35% glycolic acid (or glycolide)/65% lactic acid (orlactide) to 10% glycolic acid (or glycolide)/90% lactic acid (orlactide), which are soluble in benzene, have an inherent viscosity at 1%in chloroform at 25° of from 0.08 to 0.5 dl/g, and have a narrow to widepolydispersity.

[0113] Continuous release of peptides over a relatively longer period oftime, say 2 to 6 months, from formulations according to this invention,may be achieved using polylactic acid homopolymer or co-polyestershaving a molar composition ranging from 35% glycolic acid (orglycolide)/65% lactic acid (or lactide) to 0% glycolic acid (orglycolide)/100% lactic acid (or lactide), which are benzene-soluble,have an inherent viscosity at 1% w/v in chloroform at 25° C. of from0.08 to 0.8 dl/g, and have a narrow to wide polydispersity.

[0114] Continuous release of peptides over a very long period of time,say up to 2 years, from formulations according to this invention, may beachieved using polylactic acid homopolymer or co-polyesters having amolar composition ranging from 25% glycolic acid (or glycolide)/75%lactic acid (or lactide) to 0% glycolic acid (or glycolide)/100% lacticacid (or lactide), which are benzene-soluble, have an inherent viscosityat 1% w/v in chloroform at 25° C. of from 0.2 to 4.0 dl/g, and a normalto high polydispersity.

[0115] Timed or pulsed release (with an induction period prior torelease), or discontinuous release (where there is an initial phase ofrelease followed by a period of no release or ineffective release,followed by a second phase of release), over a relatively short periodof time, say up to 2 months, may be acheived with the formulationaccording to this invention, using benzene-insoluble polymers which havea narrow to most-probable molecular weight distribution, and an inherentviscosity at 1% w/v in chloroform at 25° C. from 0.3 to 4.0 dl/g.

[0116] Yet another feature of the present invention, which is novel anddistinguishes this invention from all other previously describedcontrolled release drug delivery system based on polyesters orco-polyesters, and which further controls the rate of release, is thelevel of incorporation of peptide as the polyester salt (optionally inthe presence of free drug and/or free polymer). This further controllingfeature differs entirely from those parameters which result in increasedrelease rates in more conventional delivery systems based on polyesters,which are directed towards the delivery of highly lipophilic drugshaving relatively low aqueous solubility, such as steroids. In thosecases, as the level of drug incorporation increases, an increased rateof release is generally seen, even though the water uptake of suchsystems is reduced, due to the increased phase volume of lipophilicdrug. In fact, such increased rates of release of drugs such as steroidsare dependent on the drug retaining its thermodynamic identity, and onsimple Fickian diffusion kinetics (see Baker and Lonsdale, loc. cit.)That is, for drugs such as steroids, as drug loading increases, andproviding the lipophilic drug has some solubility in the lipophilicpolymer, simple Fickian diffusion rates are increased.

[0117] A totally different situation exists, however, with the productsof the present invention. It is now recognised that a major componentpart of the degradation of polyesters and co-polyesters is hydrolysis ofester groups, and the rate at which this occurs is dependent on wateruptake (see Pitt and Zhong-wei Gu, J. Controlled Release, 4, 283-292(1987); Hutchinson and Furr, ibid., 13, 279-294 (1990)). Peptides arehydrophilic, and their salt formation with polyesters results in a phasecontaining polyester-drug salt which has a higher water uptake than thepolyester alone. That is, the polyester chain in the salt can degradefaster than free polyester alone, which has a similar composition,molecular weight and polydispersity. As peptide release is stronglydependent on degradation, then release is governed in part by both thelevel of incorporation of the polyester-peptide drug salt in thecomposition, and the proportion of peptide in the salt. For polyestersor co-polyesters of the same composition and structure, increasing oneor both of these parameters results in increased rates of release, andby implication can reduce, in certain circumstances, the periods of timeover which release can occur. Levels of peptide drug incorporation,either as polyester-drug salt, or as polyester-drug salt in combinationwith free peptide, preferably range from 0.1% w/w to 75% w/w in thepolyester-drug formulation.

[0118] The peptide drug loading in the composition of the invention andits variation with polyester molecular weight and polydispersity, is asfollows. For continuous release of a peptide over very long periods oftime, say up to 2 years, low levels of drug incorporation, ranging from1.0% to 20% w/w, are preferred, using polyesters which have a preferredweight average molecular weight of 20,000 Da or more andpolydispersities greater than 2.2 and preferably greater than 3.5. Theseparameters for very long term release also depend in part on otherfeatures within the drug formulation, such as composition with respectto co-monomer content, structure, solubility/insolubility in benzene,and geometry and dimensions of the dosage form. A polyester of weightaverage molecular weight of about 20000 has an inherent viscosity ofabout 0.2, dependent upon such factors as its structure, composition andpolydispersity.

[0119] For continuous release over relatively long periods of time, sayup to 6 months, preferred levels of peptide drug incorporation rangefrom 0.5% to 35% w/w, using polyesters or co-polyesters having weightaverage molecular weights of preferably 10,000 Da or more, andpolydispersities greater than 1.8 and preferably greater than 2.2,depending on all other parameters such as composition, structure,solubility/insolubility in benzene, and geometry and dimensions of thedosage forms.

[0120] For continuous release over relatively short periods of time, sayup to 2 months, preferred levels of peptide drug incorporation rangefrom 0.1% to 75% w/w, using polyesters having preferred weight averagemolecular weights of 2,000 Da or more, and polydispersities greater than1.2, depending on all other parameters such as composition, structure,solubility/insolubility in benzene, and geometry and dimensions of thedosage forms.

[0121] An additional parameter which further controls peptide drugrelease from formulations according to this invention, and which isabsent from prior art types of delivery systems based on homo- andco-polymers of lactic acid and glycolic acid, is the functionality ofthe peptide, with regard to the number of basic groups such as arginineand lysine residues in the peptide drug molecule, and the functionalityof the polyester or co-polyester with respect to the average number ofcarboxylic acid groups contained by the average polymer or co-polymerchain. In general, for continuous release of the peptide drug, thegreater the level of such polyfunctional interaction in thepeptide-polyester polyelectrolyte complex, the greater thepolydispersity required. In contrast, for discontinuous or pulsedrelease, polydispersities of less than 2.2 are preferred.

[0122] One of the relatively rare occurrences of mutual compatibility orsolubility of two polymer types having different chemical structures, isrepresented by mixtures of polyesters, based on homo- and co-polymers oflactic and glycolic acids, with low molecular weight polyoxyethylenes,and in particular low molecular weight polyethylene glycols. Thiscompatibility has been put to good effect in polyester-peptide drugsalts and their preparation, in the present invention, in a novel andunexpected way. Thus, it is known that certain pharmacologically activepeptides can be ‘pegylated’, that is conjugated with a polyethyleneglycol or alkoxy-polyethylene glycol, in such a way that thepharmacological activity of the peptide is retained. The presence in thepegylated peptide molecule of the conjugated polyoxyethylene chain thusrenders the pegylated peptide partially compatible with the polyester orco-polyester.

[0123] Thus, providing that the remaining lysine or arginine residues inthe pegylated peptide occur as salts of weak acids, this compatibilityfacilitates the preparation of the polyester-peptide drug salt, as wellas adding a further element of control of release. Pharmacologicallyactive conjugates of peptides with other water-soluble polymers, such aspolysaccharides, synthetic polypeptides and polyvinyl pyrrolidone, arealso useful, but are less preferred as none of these latterwater-soluble polymers is soluble or compatible with the polyester orco-polyester.

[0124] This invention preferably applies to pharmacologically activedrugs containing basic functionality. However, it can also be applied topeptides which are pharmacologically active and which are either neutralor tend to exist largely as polyanions (polypeptides having excesscarboxylic acid functionality).

[0125] In the first of these instances (a pharmacologically activeneutral polypeptide containing neither acidic nor basic residues) a saltof a synthetic polypeptide, which contains basic functionality and whichis pharmacologically inactive, and the polyester, is used. Such a saltof the pharmacologically inactive synthetic polypeptide and thepolyester or co-polyester is also amphipathic, and so can act as adispersing agent for solubilising or colloidally dispersing apharmacologically active, but neutral, peptide in an organic phase.

[0126] In the second of these cases, (where the pharmacologically activepolypeptide contains residual carboxylic acid functionality), a salt ofa synthetic polypeptide having at least two basic groups in thesynthetic polypeptide chain, and which is pharmacologically inactive,and a polyester or co-polyester, is used. In this second case, in thesalt of the synthetic polypeptide and polyester, the concentration ofbasic functional groups in the salt is greater than the concentration ofcarboxylic acid groups in the acidic, pharmacologically active peptide.This excess basic functionality in the salt can then interact by furthersalt formation with the carboxylic acid groups of the acidicpharmacologically active peptide. The resulting salts complex may thenbe solubilised or dispersed in an organic solvent or phase which isnormally a total non-solvent for the peptide in question, but which aresolvents for the polyester or co-polyester, in the manner describedabove for other polyester-peptide salts.

[0127] Because salts of peptides containing basic functionality withpolyesters and co-polyesters containing carboxylic acid functionalityare amphipathic, their surface active properties can be used tofacilitate the dispersion of other hydrophilic drugs, or aqueoussuspensions of such drugs, in an organic solvent or phase containing thepolyester-peptide salt. The use of such amphipathic salts of peptideswith polyesters or co-polyesters as dispersing or solubilising agentsforms a further feature of this invention.

[0128] The invention is illustrated, but not limited, by the followingExamples.

[0129] The measurement of viscosities and their relationship to thevarious averaged molecular weights are discussed in Sorensen andCampbell, “Preparative Methods of Polymer Chemistry”, 2nd edition, 1968,Interscience Division of John Wiley, pages 43-50. In the Examplesdescribed below herein, an Ubbelohde viscometer giving a flow time forchloroform alone of about 100 seconds was used. Chloroform was used asthe solvent as this was a solvent for both benzene-soluble andbenzene-insoluble polymers over the composition range disclosed.

[0130] Molecular weights and molecular weight distributions ofpolyesters described in this application of molecular weight greaterthan about 2000 Da, were determined by size exclusion chromatography,relative to polystyrene standards, using 3×30 cm PL Gel, 10 μm mixed Bcolumns (ex Polymer Laboratories, Church Stretton, Shropshire, UK)connected in series and fitted with a 10 μm guard column.Tetrahydrofuran was used as solvent at 40° C. with a nominal flow rateof 1 ml per minute. Molecular weight characteristics were calculatedusing Data Analysis Package Perkin-Elmer 7700 Professional Computer withGPC software.

[0131] For measurement of molecular weights of less than 2000 Da, sizeexclusion chromatography is not the preferred method of molecular weightdetermination, and instead non-aqueous potentiometric titration can beused, to give either the molecular weight or equivalent weight of thepolyester, by direct measurement of the carboxylic acid content of thepolyester or co-polyester. Non-aqueous potentiometric titrations weregenerally carried out using a known weight of polyester or co-polyesterdissolved in acetone containing 10% v/v of water. Titrations werecarried out using dilute sodium hydroxide solutions and using equipmentsupplied by Radiometer (Copenhagen, Denmark). This consisted of atitrator (TTT 80) and autoburette (ABU 80), a pH meter (PHM 83) and aRussell CMAWK electrode. The titration was plotted on a Servograph (REC80) and molecular weight of the polymer is$\frac{w \times 1000 \times f}{v \times n}$

[0132] where w is the weight of polymer used,

[0133] f is the average number of carboxylic acid groups per polymerchain

[0134] v is the volume of sodium hydroxide used,

[0135] n is the normality of the sodium hydroxide used.

EXAMPLE 1

[0136] Goserelin acetate (100.6 mg, equivalent to about 86 mg of peptideas free base), and 50/50% molar D,L-lactide/glycolide co-polymer (300.3mg) containing one terminal carboxylic acid group per polymer chain andhaving a weight average molecular weight of 4300 Da and an inherentviscosity at 1% w/v in chloroform at 25° C. of 0.08 dl/g, and which wasinsoluble in benzene, were dissolved in anhydride-free glacial aceticacid (3 ml). The acetic acid solution of drug and polymer was addeddropwise to liquid nitrogen, and the frozen droplets were freeze-driedfor 24 hours under high vacuum conditions. The freeze-dried product wasfinally post-dried at 50° C. for 24 hours under high vacuum, to give apolyester-drug mixture containing nominally about 25% w/w goserelinacetate (equivalent to about 22.3% w/w peptide as free base).

[0137] The dried polyester-drug mixture (400 mg) was added todichloromethane, and made up to 4 ml. Initially, a cloudy colloidalmixture was obtained, but over the course of 1 hour this graduallycleared to form a clear solution. This solution was cast as a film, andallowed to dry at room temperature for about 6 hours, then for 20 hoursat 50° C. under high vacuum. A clear, transparent film containingpolyester-drug salt was thus obtained.

[0138] (i) The clear, transparent film (100 mg) thus obtained was meltedand compression moulded at 80° C. to give a transparent film, about 0.02cm thick. On immersion in water at 37° C. for 24 hours, the weight ofthe hydrated drug/polymer film increased to 225 mg. In contrast, thepolyester alone (100 mg) similarly treated increased in weight to only126 mg, and a film comprising a simple admixture of goserelin acetate(25 mg) and polymer (75 mg) (made by adding drug to a solution ofpolymer in dichloromethane, removing the solvent and compressionmoulding the resulting material to give film about 0.02 cm thick)weighed only 136 mg after 24 hours immersion in water at 37° C. It isapparent from this experiment that the polyester-drug salt compositionis considerably more hydrophilic, and has a higher water-uptake, thaneither the polyester alone or simple admixtures of drug and polyester.

[0139] In the simple admixture of drug and polymer in dichloromethane,the drug showed no sign of dissolving even after I month, and when driedand compression moulded, the simple admixture gave an opaque film.However, in a further experiment, the clear, transparent film obtainedabove (100 mg) was dissolved in dichloromethane (1 ml) to give a clear,transparent polyester-drug solution. To this solution was addedtrifluoroacetic acid (50 μl), and the mixture was stirred vigorously.There was an immediate precipitate of goserelin as the trifluoroacetatesalt.

[0140] These two experiments show that the clear, transparent filmcontaining polyester-drug salt, obtained as described above, is capableof being processed to a shaped delivery system using conventionalpolymer melt fabrication techniques. Further, this product containsvirtually no acetic acid or acetate anion, and so the drug must exist inthe form of the polyester salt. The polyester-drug salt arises becausethe terminal lactic or glycolic acid groups-on the co-polymer are muchstronger acids than acetic acid, and so the weaker acetic acid isdisplaced by the polymer. The polymer carboxylic acid in thedichloromethane-soluble polyester-drug salt can in turn be displaced bya very much stronger carboxylic acid, such as trifluoroacetic acid. Whenthis occurs, the trifluoroacetate salt of the peptide is formed and, asit is not soluble in dichloromethane, is precipitated.

[0141] (ii) The clear, transparent film obtained as described above (50mg), containing the polyester-drug salt, was moulded to give a filmabout 0.02 cm thick. The film was incubated in phosphate buffered saline(containing 0.02% sodium azide) at pH 7.4 and 37° C., and the buffersolution was assayed periodically by UV to determine the amount ofgoserelin released. This moulded product released goserelin continuouslyover about 2 weeks, and by 3 weeks had virtually degraded completely,and disappeared from the incubation medium.

[0142] This experiment demonstrates the utility of very low molecularweight, benzene-insoluble polymers for delivery of drug over a shorttime interval.

[0143] Similar moulded formulations can be manufactured using, in placeof goserelin acetate, either naturally occuring gonadotrophin releasinghormones or other highly potent synthetic analogues (agonistic orantagonistic) of gonadotrophin releasing hormone, such as tryptorelin,leuprorelin, buserelin and nafarelin, preferably as the acetate salts orsalts with other weak acids; or any other polypeptide hormone whichcontrols secretion of the intact gonadotrophin or either of thegonadotrophin subunits.

EXAMPLE 2

[0144] The clear, transparent film product obtained in Example 1 above(100 mg) and a 50/50 molar D,L-lactide/glycolide co-polymer (1.05 g)having a weight average molecular weight of 121,000 Da and an inherentviscosity at 1% w/v in chloroform at 25° C. of 0.84 dl/g, and which isinsoluble in benzene, were dissolved in dichloromethane (100 ml). Thesolution was stirred vigorously at 1000 revolutions per minute (rpm),and silicone oil (50 ml) was added slowly over 1 hour, to precipitateboth the polyester-drug salt and the free polyester. After 1 hour, thepartially precipitated mixture of polyester-drug salt, free polyester,silicone oil and dichloromethane was added to vigorously stirred hexane(2 liters) to harden the microparticles of polyester-drug salt and freepolyester. This mixture was stirred for 2 hours and then allowed tosettle, and the hexane layer was discarded. The microparticles(containing about 1.95% w/w goserelin as free base) were washed threetimes with fresh hexane (500 ml), and finally isolated by filtration anddried at 35° C. for 24 hours under high vacuum. The average size of theapproximately spherical microparticles so obtained, which comprise asolution of polyester-drug salt in free polymer, was about 30 μm.

[0145] A portion of this product (250 mg) was incubated inphosphate-buffered saline (containing 0.02% sodium azide) at pH 7.4 and37° C., and the buffer solution was assayed periodically by UV todetermine the amount of goserelin released. The microparticles releaseddrug over about 5 weeks, and by 7 weeks had virtually disappeared fromthe incubation medium.

[0146] The polymer composition used in this experiment was a mixture oftwo co-polymers of the same lactide/glycolide composition, but havingwidely different molecular weights, and which as a mixture, as describedhere, was insoluble in benzene, had a weight average molecular weight of108,000 Da, a polydispersity of 5.1, and an inherent viscosity at 1% w/vin chloroform at 25° C. of 0.72 dl/g.

[0147] These experiments show the utility of benzene-insolublepolyesters having a high molecular weight and a high polydispersity, forrelease of goserelin over relatively short periods of time of 5-7 weeks.

[0148] Similar microparticle formulations can be manufactured using, inplace of goserelin acetate, either naturally occurring analogues ofgonadotrophin releasing hormones or other highly potent syntheticanalogues (agonists or antagonists) of gonadotrophin releasing hormone,such as tryptorelin, leuprorelin, buserelin or nafarelin, preferably asthe acetate salts or salts with other weak acids; or any otherpolypeptide hormone which controls or modulates secretion of the intactgonadotrophins or either of the individual gonadotrophin sub-units.

EXAMPLE 3

[0149] Goserelin acetate (101 mg, equivalent to about 86 mg of goserelinas free base) and a 100% molar poly(D,L-lactic acid), (299.7 mg), whichwas soluble in benzene, had a weight average molecular weight of about5400 Da, an inherent viscosity at 1% w/v in chloroform at 25° C. of 0.08dl/g, and a polydispersity of 1.8, were dissolved in anhydride-freeglacial acetic acid (4 ml). This acetic acid solution of goserelin andpolyester was added dropwise to liquid nitrogen, and the frozen dropletswere isolated, freeze-dried under vacuum for 24 hours, and then dried at55° C. for 24 hours under high vacuum.

[0150] (i) The resulting dried product was added to dichloromethane (4ml), to give a cloudy mixture initially, which rapidly dissolved to givea clear solution which was filtered through a 0.2 μm nylon sterilisingfilter.

[0151] This experiment shows that solutions of the polyester salt ofgoserelin can be sterile-filtered, in contrast to mixtures ordispersions of simple drug salts in an organic solution of thepolyester.

[0152] (ii) Trifluoroacetic acid (50 μl) was added to the cleardichloromethane solution from (i) above (1 ml), with vigorous agitation.There was an immediate precipitation of goserelin as itstrifluoroacetate salt, showing that the goserelin was present in thedichloromethane solution as the salt with the carboxy-terminatedpolyester.

[0153] Similar sterile solution formulations can be manufactured using,in place of goserelin acetate, either naturally occurring gonadotrophinreleasing hormones or other highly potent synthetic analogues (agonisticor antagonistic) of gonadotrophin releasing hormone, such astryptorelin, leuprorelin, buserelin or nafarelin, preferably as theacetate salts or salts with other weak acids; or any other polypeptidehormone which controls or modulates secretion of the intactgonadotrophins or either of the individual gonadotrophin—sub-units.

EXAMPLE 4

[0154] The dichloromethane solution of goserelin-polyester obtained inExample 3 (2 ml) was diluted with more dichloromethane and made up to 10ml. This solution was sprayed into vigorously stirred hexane (1 liter),to give microparticles which, after isolation and drying under vacuum at45° C. for 24 hours, ranged in size from about 2 μm to about 30 μm, withan average size of about 10 μm. The goserelin content of thesemicroparticles was equivalent to about 22% as free base.

[0155] These microparticles were incubated in saline, buffered withphosphate to pH 7.4 at 37° C., and the supernatant periodically assayedby UV for goserelin. Goserelin was released continuously, the releasewas essentially complete by about 8 weeks, and by 11 weeks themicroparticles had totally degraded and disappeared from the incubationmedium. This experiment shows the utility of very low molecular weightbenzene-soluble polyesters in providing continuous peptide release overabout 2 months.

[0156] If the goserelin acetate in the above experiments is replaced bythe trifluoroacetate salt, then a clear solution is not obtained, butinstead the polyester solution in dichloromethane contains essentially adispersion of goserelin trifluoroacetate. This mixture will not passthrough a 0.2 μm filter, and so is not capable of beingsterile-filtered; and such a dispersion of goserelin trifluoroacetate inthe polyester solution, when sprayed into stirred hexane produced acongealed and flocculated mass, rather than microparticles.

[0157] Thus the goserelin-polyester salt has properties which render itmuch easier to formulate into a microparticle form, than mixtures of thesimple salt in a solution of very low molecular weight polymer.

[0158] Similar microparticle formulations may be manufactured by using,in place of goserelin acetate, either naturally occurring gonadotrophinreleasing hormones or other highly potent synthetic analogues (agonistsor antagonists) of gonadotrophin releasing hormone, such as tryptorelin,leuprorelin, buserelin or nafarelin, preferably as the acetate salts orsalts with other weak acids; or any other polypeptide hormone whichcontrols or modulates secretion of the intact gonadotrophins or eitherof the individual gonadotrophin sub-units.

EXAMPLE 5

[0159] Goserelin acetate (304 mg, equivalent to about 248 mg ofgoserelin as free base) and 100% molar poly(D,L-lactic acid) (102 mg),having a weight average molecular weight of about 5400, an inherentviscosity at 1% w/v in chloroform at 25° C. of 0.08 dl/g, and apolydispersity of 1.8, were dissolved in anhydride-free glacial aceticacid (2 ml). The acetic acid solution of goserelin and polyester wasthen added dropwise to liquid nitrogen, and the frozen droplets wereisolated, freeze-dried under high vacuum for 24 hours, and then driedunder vacuum at 55° C. for 24 hours.

[0160] The resulting product was added to dichloromethane (2 ml) to givea cloudy, colloidal mixture which did not clear totally with time. Thismixture in dichloromethane comprised essentially a dispersion ofgoserelin acetate in the goserelin-polyester salt.

[0161] This dispersion of goserelin acetate in the methylene chloridesolution of the polyester-goserelin salt was formulated into amicroparticulate form, containing goserelin equivalent to about 72% w/was free base, wherein the free goserelin acetate is dispersed throughouta continuous phase of the goserelin-polyester salt, by spray drying,spray-congealing, simple precipitation or by phase separationco-acervation.

[0162] Similar microparticle formulations may be manufactured by using,in place of goserelin acetate, either naturally occuring gonadotrophinreleasing hormones or other highly potent synthetic analogues (agonistsor antagonists) of gonadotrophin releasing hormones, such astryptorelin, leuprorelin, buserelin or nafarelin, preferably as theacetate salts or salts with other weak acids; or any other polypeptidehormone which controls or modulates secretion of the intactgonadotrophins or either of its individual sub-units.

EXAMPLE 6

[0163] A co-polyester of D,L-lactic acid and glycolic acid, having amolar composition of 78% D,L-lactic acid and 22% glycolic acid, wasprepared by co-polycondensation of the two hydroxy acids. Afterpurification of the co-polymer, by addition of a solution of theco-polyester in acetone to methanol to precipitate the co-polyester, andseparation and drying the precipitated material, the co-polyester had aweight average molecular weight of about 11,000 Da, a number averagemolecular weight (as determined by non-aqueous potentiometric titrationand assuming that each co-polyester chain has only one terminalcarboxylic acid group) of 6100 Da, and therefore a polydispersity of1.6, and an inherent viscosity at 1% w/v in chloroform at 25° C. of 0.15dl/g.

[0164] Goserelin acetate (228.9 mg, equivalent to about 200 mg ofgoserelin as free base) and the above-described co-polyester (1.8 g)were dissolved in anhydride-free glacial acetic acid (10 ml). Thegoserelin-polyester solution so obtained was added dropwise to liquidnitrogen, and the frozen droplets were isolated, freeze-dried for 24hours, and then finally dried at 50° C. for 24 hours under vacuum.

[0165] The dried goserelin-polyester mixture was added todichloromethane (10 ml) to give initially a cloudy colloidal mixture,but after 24 hours this had changed to a clear solution, which could befiltered through a 0.2 μm nylon sterilising filter.

[0166] When trifluoroacetic acid was added to a small aliquot of thisclear solution, there was an immediate precipitate of the goserelin asits trifluoroacetate salt, showing that, in the clear, transparentdichloromethane solution, the goserelin in the goserelin-polyestermixture was present mainly or wholly as the polyester salt.

[0167] The dichloromethane solution of the goserelin-polyester salt wasevaporated to dryness, and the resulting solid was dried at roomtemperature for 6 hours and then at 55° C. for 20 hours under vacuum, togive a clear cast film containing goserelin-polyester salt.

[0168] The dried goserelin-polyester mixture, prepared as describedabove, (1 g) was dissolved in 8 ml of dichloromethane. The resultingsolution was placed in a 250 ml multinecked round-bottomed flask andswept with a stream of nitrogen to remove all air, and to generate acarbon dioxide-free atmosphere. Water (90 ml), which had previously beendegassed to remove all carbon dioxide and then stored under carbondioxide-free nitrogen, was introduced into the flask, and the mixturewas stirred vigorously at about 500 rpm under an atmosphere which wasessentially carbon dioxide-free. The dichloromethane solution ofgoserelin-polyester salt rapidly dispersed to give a stable oil(dichloromethane solution of drug-polymer salt)-in-water dispersion.Whilst maintaining stirring at about 200 rpm, a vacuum was graduallyapplied and the bulk of the dichloromethane was evaporated under vacuum,to give a dispersion of goserelin-polyester salt in water. Freeze-dryingthis dispersion produced microparticles, in which the goserelin ispresent as the goserelin-polyester salt having an average particle sizeof about 20 μm, which was shown to release goserelin over about 6 weeks,when incubated in saline, buffered with phosphate to pH 7.4 at 37° C.,and the supernatant periodically assayed by UV for goserelin.

[0169] Similar microparticles may also be manufactured by incorporatingin the aqueous phase agents which are known to improve polypeptidestability such as mannitol. Although it is preferred to carry out theabove process in a carbon dioxide-free atmosphere, it is neverthelesspossible to achieve satisfactory results in the presence of traces ofcarbon dioxide, depending on polyester molecular weight and drugloading.

[0170] Similar sterile solution, cast film and microparticleformulations may be manufactured in a similar manner using, in place ofgoserelin acetate, either the natural analogues of gonadotrophinreleasing hormones or other highly potent synthetic analogues (agonistsor antagonists) such as tryptorelin, leuprorelin, buserelin ornafarelin, preferably as acetate salts or salts with other weak acids;or any other polypeptide hormone which can control or modulate thesecretion of intact gonadotrophins or either of their sub-units.

EXAMPLE 7

[0171] The procedure described in Example 5 was repeated, to give theclear transparent film, and this film (1 g) was dissolved indichloromethane (4 ml). The solution was warmed to about 35° C., andthen an aqueous solution, at about 40° C., of purified gelatin (15 mg)in water (100 μl) was added to the dichloromethane solution ofgoserelin-polyester salt, and the mixture was stirred vigorously atabout 35° C. to give an extremely fine dispersion of the aqueous gelatinsolution in the dichloromethane solution of the goserelin-polyestersalt. On cooling to room temperature, the colloidal nature of thesuspension was maintained.

[0172] This experiment demonstrates that the goserelin-polyester salthas surface active properties, and can be used to give stabledispersions in an oily phase, such as dichloromethane, of aqueoussolutions of other water-soluble agents, such as gelatin,polysaccharides and other hydrophilic polymers, or vice versa.

[0173] The process described in Example 6 was repeated, using thedispersion of aqueous gelatin in the dichloromethane solution of thegoserelin-polyester salt described above, to give a microcapsule productwhich contains both gelatin and goserelin-polyester salt.

[0174] Other low molecular weight compounds may be incorporated in theaqueous polymer phase. In particular, it is sometimes useful to includecompounds such as mannitol, which are known to enhance the stability ofpeptides. Alternatively, these stabilising agents may be incorporated inboth aqueous phases of the complex water-in-oil-in-water dispersion,comprising aqueous gelatin dispersed in the dichloromethane solution ofthe goserelin-polyester salt, and the resulting water-in-oil dispersionin turn is dispersed in water.

[0175] Similar suspension and microparticle formulations may bemanufactured similarly using, in place of goserelin acetate, otherhighly potent analogues (agonists or antagonists) of gonadotrophinreleasing hormone, such as tryptorelin, leuprorelin, buserelin ornaferelin, preferably as the acetate salts or salts with other weakacids; or any other polypeptide hormone which can control or modulatethe secretion of intact gonadotrophins or either of their sub-units.

EXAMPLE 8

[0176] Goserelin acetate (771 mg, equivalent to about 670 mg ofgoserelin as free base), 95/5 molar D,L-lactide/glycolide co-polymer(1.8 g) having a weight average molecular weight of about 3600 Da and aninherent viscosity at 1% w/v in chloroform at 25° C. of 0.08 dl/g, and95/5 molar D,L-lactide/glycolide co-polymer having a weight averagemolecular weight of about 15,000 Da and an inherent viscosity at 1% w/vin chloroform at 25° C. of 0.17 dl/g (4.2 g), were dissolved inanhydride-free glacial acetic acid (70 ml). The combined polymers had aweight average molecular weight of about 12,300 Da and a polydispersityof about 2.6. The goserelin-polyester solution was added dropwise toliquid nitrogen, and the frozen droplets were isolated and freeze-driedunder high vacuum for about 18 hours. The product drug-polymer mixturewas finally dried at 55° C. for 24 hours under high vacuum.

[0177] The dried drug-polymer mixture (6 g) was added to dichloromethane(60 ml) to give an initially cloudy colloidal mixture which, over thecourse of 1 hour, gradually cleared to give a clear solution ofgoserelin-polyester salt in dichloromethane.

[0178] This solution was spray-dried using a Buchi spray dryer, using aninlet temperature of 60° C. and an outlet temperature of 35° C., toproduce approximately spherical microparticles of about 1 μm to about 1μm diameter.

[0179] In these microparticles the drug is present essentiallycompletely as the goserelin-polyester salt, as the acetic acid content,as free acid or anion, is 0.06% or less, instead of 0.6 to 0.7% whichwould be required if the goserelin were present as its acetate salt.

[0180] These microparticles when further processed by compressionmoulding at 80° C. yielded a clear, transparent and brittle film.

[0181] This experiment demonstrates the utility of peptide salts withbenzene-soluble polyesters of low molecular weight polymers, andoptionally of high polydispersity.

[0182] Similar solution, microparticle and moulded formulations may bemanufactured using, in place of goserelin acetate, either naturallyoccurring gonadotrophin releasing hormones or other highly potentsynthetic analogues (agonists or antagonists) of gonadotrophin releasinghormone, such as tryptorelin, leuprorelin, buserelin or nafarelin,preferably as the acetate salts or salts with other weak acids; or anyother polypeptide hormones which controls secretion of the intactgonadotrophins or either of the gonadotrophin sub-units.

EXAMPLE 9

[0183] Goserelin acetate and other highly potent synthetic agonists ofgonadotrophin releasing hormone are selective chemical castrating agentswhich are used in the treatment of hormone dependent cancers such asprostate cancer in men and premenopausal breast cancer in women. Thesedrugs are also used to treat non-malignant gynaecological conditions inwomen, and they work by ultimately suppressing the secretions ofgonadotrophins by the pituitary, which in turn leads to a suppression ofthe sex hormones, such as oestrogen in females and testosterone inmales.

[0184] Consequently, continuous sustained release of such drugs may beevaluated in vivo in the normal adult female rat having regular oestruscycles. In this animal, the oestrus cycle is about 4 Days, and theoccurrence of oestrus is shown by the presence of only cornified cellsin vaginal smears, taken on the day of oestrus. If the animal ischemically castrated, by a drug such as goserelin, then oestrous doesnot occur, leading to the absence of cornified cells in vaginal smears.The animals will enter a prolonged period of dioestrous, induced bychemical castration, and dioestrous will be maintained for as long aseffective amounts of drug are released.

[0185] (i) The microparticles obtained in Example 8 (450 mg) weredispersed in water containing 2% w/v of sodium carboxymethyl celluloseand 0.2% w/v polysorbate 80, and made up to 3 ml with water. 0.2 ml(equivalent to about 3 mg of goserelin as free base) was injectedsub-cutaneously into 10 normal adult female rats showing regularcyclicity, and the ensuing effect on oestrous cyclicity was determinedby microscopic examination of vaginal smears. The animals entered acontinuous phase of dioestrous, that is chemical castration, lasting95±3 Days.

[0186] This experiment shows that an aqueous suspension formulation ofgoserelin-polyester salt, based on a low molecular weightbenzene-soluble polyester, provides a relatively long period ofcontrolled release of about three months of a peptide drug which has ametabolic half-life of only 4-6 hours.

[0187] (ii) The microparticles obtained in Example 8 (450 mg) weredispersed in ethyl oleate, and made up to 3 ml. Again 0.2 ml offormulation were administered to (six) female rats showing regularlycyclicity by subcutaneous injection. The animals entered a continuousphase of dioestrous lasting 81±3 Days.

[0188] This experiment shows that a solution formulation ofgoserelin-polyester salt in an organic injection vehicle, which is anon-solvent for the polyester alone, provides a relatively long periodof controlled peptide drug release.

EXAMPLE 10

[0189] Leuprorelin acetate (50.3 mg) and the co-polyester comprising 78%molar D,L-lactic acid and 22% molar glycolic acid, described in Example6 above (453.2 mg), were dissolved in anhydride-free glacial acetic acid(5 ml). The resulting solution was added dropwise to liquid nitrogen,and the frozen droplets were freeze-dried under high vacuum for 22hours, and then further dried at 55° C. for 24 hours under high vacuum.

[0190] The resulting product (500 mg) was dissolved in redistilledacetone (10 ml) in a 100 ml round bottomed flask, to give initially aturbid, colloidal mixture, which gradually cleared to a transparentsolution. The acetone was evaporated under vacuum, and the resultingclear film was dried at 55° C. for 4 hours under high vacuum. This filmof leuprorelin-polyester salt was redissolved in acetone (10 ml), andthe solution was degassed and then purged with nitrogen.

[0191] Freshly distilled water (200 ml) was stirred vigorously undernitrogen, and the acetone solution of leuprorelin-polyester salt wassprayed onto the surface of the agitated water. When all the acetonesolution had been sprayed, stirring was maintained for a further hour,and then the mixture was allowed to settle. The microparticles of theleuprorelin-polyester salt settled out, and the aqueous supernatant wasdiscarded. The microparticles were resuspended in a further portion ofcarbon-dioxide free water (^(˜)200 ml), and the suspension was stirredunder nitrogen for a further hour. The microparticles were separated, byinitially allowing the mixture to settle, decanting the aqueous layer,and then filtering the residue to separate the microparticles from theexcess water. The microparticles were dried at 30° C. for 24 hours underhigh vacuum, to give a product which had an average particle size ofabout 15 μm.

[0192] This microparticle formulation of leuprorelin-polyester salt wasincubated in saline, buffered with phosphate to pH 7.4 at 37° C., andthe supernatant was assayed periodically by UV for leuprorelin.Leuprorelin was released continuously for about 5 weeks, by which timethe formulation had totally degraded.

[0193] Similar microparticle formulations may be manufactured similarlyusing, in place of leuprorelin, either naturally occurring gonadotrophinreleasing hormones or other highly potent synthetic analogues (agonistsor antagonists) of gonadotrophin releasing hormone, such as tryptorelin,goserelin, buserelin or nafarelin, preferably as the acetate salts orother salts with weak acids; or any other polypeptide hormones whichcontrols secretion of the intact gonadotrophins or either of thegonadotrophin sub-units.

EXAMPLE 11

[0194] i) Goserelin acetate (2.28 g, equivalent to about 2.00 g ofgoserelin as free base) was dissolved in anhydride-free glacial aceticacid (60 ml). A mixture of two 95/5% molar poly(D,L-lacticacid)/polyglycolic acid) copolymers (12.6 g of a copolymer with a weightaverage molecular weight of 15,846 and a polydispersity of 1.38, and 5.4g of a copolymer with a weight average molecular weight of 3,896 and apolydispersity of 1.78) and therefore providing an excess of copolymercarboxylic acid end groups relative to basic drug, was dissolved withstirring in anhydride-free glacial acetic acid (150 ml) to give a clearsolution. The drug solution was added to the copolymer solution and wasmixed thoroughly. This mixture was then added dropwise to liquidnitrogen to freeze it as small beads, and the solid material was freezedried for two days using an Edwards high vacuum freeze drier. The driedmaterial was further dried at 50-55° C. in a vacuum oven for 24 hours.

[0195] This dried product (100 mg) was added to dichloromethane (1 ml)and was found to dissolve totally within 2 hours to give a clearsolution. It is shown by this Example that the formation of thepolyester-goserelin salt confers good solubility upon the drug such thatit can be dissolved in a non-polar solvent.

[0196] ii) Goserelin acetate (2.28 g, equivalent to about 2.00 g ofgoserelin as free base) was dissolved in anhydride-free glacial aceticacid (60 ml). A mixture of two 100% molar poly(D,L-lactic acid) polymers(12.6 g of a polymer with a weight average molecular weight 15,178 and apolydispersity of 1.27, and 5.4 g of a polymer with a weight averagemolecular weight of 4,204 and a polydispersity of 1.84) and thereforeproviding an excess of copolymer carboxylic acid end groups relative tobasic drug, was dissolved with stirring in anhydride-free glacial aceticacid (150 ml) to give a clear solution. The drug solution was added tothe polymer solution and was mixed thoroughly, and this mixture was thenadded dropwise to liquid nitrogen to freeze it as small beads. The solidmaterial was freeze dried for two days using an Edwards high vacuumfreeze drier, and the dried material was further dried at 50-55° C. in avacuum oven for 24 hours.

[0197] This dried product (10 mg) was added to dichloromethane (1 ml)and was found to dissolve totally within 2 hours to give a clearsolution. It is shown by this Example that the formation of thepolyester-goserelin salt confers good solubility upon the drug such thatit can be dissolved in a non-polar solvent.

[0198] iii) Goserelin acetate (2.28 g, equivalent to about 2.00 g ofgoserelin as free base) was dissolved in anhydride-free glacial aceticacid (60 ml). A mixture of an 80/20% molar poly(D,L-lacticacid)/polyglycolic acid) copolymer (12.6 g of a copolymer with a weightaverage molecular weight 106,510 and a polydispersity of 2.27) and a95%/5% molar poly(D,L-lactic acid)/polyglycolic acid) copolymer (5.4 gof a copolymer with a weight average molecular weight 3,896 and apolydispersity of 1.78) and therefore providing an excess of copolymercarboxylic acid end groups relative to basic drug, was dissolved withstirring in anhydride-free glacial acetic acid (150 ml) to give a clearsolution. The drug solution was added to the copolymer solution and wasmixed thoroughly. This mixture was then added dropwise to liquidnitrogen to freeze it as small beads, the solid material was freezedried for two days using an Edwards high vacuum freeze drier, and thedried material was further dried at 50-55° C. in a vacuum oven for 24hours.

[0199] This dried product (100 mg) was added to dichloromethane (1 ml)and was found to dissolve totally within 2 hours to give a clearsolution. It is shown by this Example that the formation of thepolyester-goserelin salt confers good solubility upon the drug such thatit can be dissolved in a non-polar solvent. iv) Goserelin acetate (2.17g, equivalent to about 1.90 g of goserelin as free base) was dissolvedin anhydride-free glacial acetic acid (60 ml). A mixture of two 67/33%molar poly(D,L-lactic acid)/polyglycolic acid) copolymers (12.0 g of acopolymer with a weight average molecular weight of 35,833 and apolydispersity of 1.83, and 5.15 g of a polymer with a weight averagemolecular weight of 4,116 and a polydispersity of 1.86) and thereforeproviding an excess of polymer carboxylic acid end groups relative tobasic drug, was dissolved with stirring in anhydride-free glacial aceticacid (150 ml) to give a clear solution. The drug solution was added tothe copolymer solution and was mixed thoroughly. This mixture was thenadded dropwise to liquid nitrogen to freeze it as small beads. The solidmaterial was freeze dried for two days using an Edwards high vacuumfreeze drier, and the dried material was further dried at 50-55° C. in avacuum oven for 24 hours.

[0200] This dried product (100 mg) was added to dichloromethane (1 ml)and was found to dissolve totally within 10 minutes to give a clearsolution. It is shown by this Example that the formation of thepolyester-goserelin salt confers good solubility upon the drug, suchthat it can be dissolved in a non-polar solvent.

COMPARATIVE EXAMPLE

[0201] Goserelin acetate (2.28 g, equivalent to about 2.00 g ofgoserelin as free base) was dissolved in anhydride-free glacial aceticacid (60 ml). A 50/50% molar poly(D,L-lactic acid)/polyglycolic acid)copolymer (18.0gm polymer with a weight average molecular weight 22,307and a polydispersity of 2.07) and therefore providing an approximatelystoichiometric equivalent of copolymer carboxylic acid end groupsrelative to basic drug, was dissolved with stirring in anhydride-freeglacial acetic acid (150 ml) to give a clear solution. The drug solutionwas added to the copolymer solution and was mixed thoroughly. Thismixture was then added dropwise to liquid nitrogen to freeze it as smallbeads. The solid material was freeze dried for two days using an Edwardshigh vacuum freeze drier, and the dried material was further dried at50-55° C. in a vacuum oven for 24 hours.

[0202] This dried product (100 mg) was added to dichloromethane (1 ml)and was found not to have dissolved totally after 4 hours, but diddissolve to form a clear solution after 4 Days. It is shown by thisExample that the formation of the polyester-goserelin salt, to confergood solubility upon the drug such that it can be dissolved in anon-polar solvent, occurs more readily when the copolymer carboxylicacid end groups are present in excess relative to the basic drug.

[0203] The dried products i-iv were dissolved in dichloromethane andspray dried using a Buchi 190 lab scale spray drier, according to thefollowing table: Ratio product Inlet temp Outlet temp Product to solvent% ° C. ° C. i 10 48 32 ii 10 58 38 iii 2 58 44 iv 10 55 35

[0204] The spray drying of products i-iv gave small particles with adiameter approximately 1-10 μm in size as determined by scanningelectron microscopy. The final particles were assayed for acetic acidcontent using a gas chromatography assay with a limit of detection ofapproximately 0.03%. No acetic acid was found in these formulationsusing this assay and this demonstrates that the drug is present as thepolyester salt and not the acetate salt, since acetic acid levels ofapproximately 0.5% would be expected for the acetate salt.

[0205] Spray dried particles (50 mg) i-iv above were dissolved indichloromethane (0.5 ml) to give a clear solution. One drop oftrifluoroacetic acid was added to each, and in each case this resultedin the formation of a white precipitate. The samples were centrifuged tocollect the precipitates, which were washed with dichloromethane. HPLCanalysis showed the precipitated material to be goserelin. TheseExamples show that the drug can be displaced from the drug-polyestersalt in solution in a non-polar solvent by the addition of a strongacid, and that this causes the solubility properties of the drug innon-polar solvent to return to that expected of the acid salt of apeptide drug (i.e. not soluble).

EXAMPLE 12

[0206] The spray dried particles i-iv in Example 11 were dispersed (18%w/v) in an aqueous vehicle suitable for injection (2% sodiumcarboxymethylcellulose [Fluka, medium viscosity], 0.2% polysorbate 80[Tween (trade mark), Fluka].

[0207] The spray dried particles from Example 11, dispersed in theinjection vehicle described above, were injected into ten femaleWistar-derived rats. Blood samples were taken from the tails of fiverats on days 7, 14 and 28, and these samples were assayed for goserelinusing a radioimmunoassay with known specificity for the drug and provenlack of cross reactivity to metabolites.

[0208] The results of these experiments showed that this formulationachieved measurable blood levels of goserelin for at least 4 weeks.

EXAMPLE 13

[0209] Spray dried product ii of Example 11 was dispersed in thefollowing aqueous vehicles for injection.

[0210] a. sodium carboxymethyl cellulose (medium viscosity grade, Fluka)1.0%, and polysorbate 80 (Tween) 0.75%.

[0211] b. methyl cellulose (15 mPa.s, Fluka) 0.75% and polysorbate 80(Tween) 0.75%.

[0212] These formulations dispersed well in these vehicles, and weresuitable for parenteral administration.

EXAMPLE 14

[0213] Spray dried product ii of Example 11 (400 mg) was dissolved indichloromethane (4 ml). This was added, using a syringe, to a solutionof 0.25% polyvinyl alcohol (PVA) in water (Aldrich, 75% hydrolysed,molecular weight 2000) which was being stirred at 2500 rpm. After twominutes the rate of stirring was reduced to 800 rpm, stirring wascontinued for a further 30 minutes. Stirring was then stopped, and theparticles formed were allowd to settle out. The PVA solution wasdecanted and the particles were then washed twice with ice cold waterand recovered by centrifugation. The particles were finally dried byfreeze drying, and the final product was a fine particulate materialcontaining goserelin.

EXAMPLE 15

[0214] Spray dried formulation iv of Example 11 was extruded at 82° C.to give a cylindrical extrudate approximately one millimeter indiameter. This extrudate was cut to lengths weighing approximately 36 mgand containing approximately 3.6 mg of goserelin. This extrudate wascompletely clear to light rather than being of a white appearance, thelatter appearance being typical of a simple mixture of drug and polymerproduced without forming the salt of the peptide with the polyester (asin for example the commercially available ‘Zoladex’ depot-‘Zoladex’ is atrade mark). The clarity of this extrudate indicates that the peptidegoserelin is compatible with the polyester phase, rather than being in aseparate phase, which results in light scattering and a whiteappearance. This compatibility can only occur if the peptide is in thesame phase as the-polymer, i.e. it is present as the salt of thepolyester.

[0215] Single such 3.6 mg depots were implanted into 21 Wistar-derivedrats under anaesthesia. At subsequent time points groups of threeanimals were killed and the depots were retrieved. The recovered depotswere dissolved in glacial acetic acid in a volumetric flask and thepolymer was precipitated by addition of an excess of water. This wasthen filtered (Millex 0.5 μm) and the filtrate assayed for drug contentby HPLC. The release profile of the depots was calculated by referenceto the drug content of depots which had not been implanted, and whichwere included in the same assay. These depots of drug-polyester saltgave sustained release of goserelin in vivo for a period of at leastfour weeks.

EXAMPLE 16

[0216] (i) Lactide/glycolide copolymer (95/5) with a single terminalcarboxylic acid group (8.87 g, Mw=5750, polydispersity=1.5, molecularweight by end group titration=2516 g/mole, inherent vicosity at 1% w/vin chloroform=0.10 dl/g) was dissolved in dichloromethane (50 ml) withstirring. To this was added 1.13 g goserelin acetate, forming a cloudysuspension. Methanol (5 ml) was added with stirring, and after 30minutes the mixture was completely clear. The solvent was then removedfrom the solution by rotary evaporation to leave a clear solid. Thissolid was redissolved in dichloromethane (50 ml) and the solvent wasagain removed by rotary evaporation. The redissolution step and solventremoval step were repeated twice more to leave a very viscous fluidwhich was dried under high vacuum to give a white foam. The foam wasbroken up and dried under vacuum for a further 24 hours at roomtemperature to give a fine amorphous solid.

[0217] (ii) The process described in i) above was repeated, using alactide/glycolide copolymer (75/25) with a single terminal carboxylicacid (8.87 g, Mw=10900, polydispersity=1.85, molecular weight by endgroup titration=3210 g/mole, inherent viscosity at 1% w/v inchloroform=0.14 dl/g), to give a fine amorphous solid.

[0218] Formulation 1

[0219] The goserelin-lactide/glycolide polymer salt from (i) above (g)was added to benzyl benzoate (99%, ex Janssen, 2 ml) and this was heatedusing a hand held hot-air gun whilst agitating the mixture until thesolid was dissolved. 110 μl of this solution formulation contained 3.6mg of goserelin.

[0220] Formulation 2

[0221] As Formulation 1, except that the solvent was a mixture (1.7 ml)of 67% benzyl benzoate (99%, ex Janssen) and 33% benzyl alcohol(anhydrous, 99%, ex Aldrich). 100 μl of this solution formulationcontained 3.6 mg of goserelin.

[0222] Formulation 3

[0223] As Formulation 1, except that the solvent was benzyl alcohol (1.7ml, anhydrous, 99%, ex Aldrich). 100 μl of this solution formulationcontained 3.6 mg of goserelin.

[0224] Formulation 4

[0225] As Formulation 1, except that the goserelin-lactide/glycolidepolymer salt from (ii) above (1 g) and 3 ml of benzyl benzoate wereused. 150 μl of this solution formulation contained 3.6 mg of goserelin.

[0226] Formulation 5

[0227] As Formulation 4, except that the solvent mixture of Formulation2 was used. 100 μl of this solution formulation contained 3.6 mg ofgoserelin.

[0228] Formulation 6

[0229] As Formulation 4, except that the solvent of Formulation 3 wasused. 100 μl of this solution formulation contained 3.6 mg of goserelin.

[0230] Biological Evaluation

[0231] Release of goserelin from the above Formulations 1 to 6 in vivowas determined by studying daily vaginal smears of dosed female rats.The normal oestrus cycle (oestrus, dioestrus, met-oestrus, pro-oestrus),can be followed from the proportions of the various cell types(leucocytic, epithelial and cornified) in the smear. If the release ofdrug from the formulations is continuous the normal oestrus cycle isinterrupted and the rats will remain in dioestrus as long as release ofthe goserelin continues.

[0232] Formulations 1-6 were dosed to groups (n=6) of regularly cyclingfemale rats at a dose of 3.6 mg goserelin per rat. A syringe fitted witha 20 guage needle was used for dosing the formulations subcutaneously.An undosed group of five rats was used as a control group. Vaginalsmears were taken daily from the rats, and examined to determine theoestrus state of the animals, and the results obtained were as follows:Average duration of dioestrus (days) Formulation number (±s.e.) 1 100 ±2.7 2 120 ± 6.3 3  69 ± 5.9 4  59 ± 1.2 5  61 ± 2.1 6  53 ± 3.7

[0233] From these results it can be seen that all six formulations gaveperiods of goserelin release in excess of 6 weeks and that formulations1 and 2 released goserelin for a period of three months or more. It canfurther be seen from these examples that the formulations of thegoserelin-polyester salt can be provided as solutions which can bereadily administered parentally using a narrow gauge needle, and thatsuch formulations are convenient for treatment of hormone dependenttumours in man.

EXAMPLE 17

[0234] Formulation 1

[0235] As Formulation 1 from Example 16.

[0236] Formulation 2

[0237] The process described in Example 16(i) was repeated, using apolylactide homopolymer with a single terminal carboxylic acid (Mw=5092,polydispersity=1.44, molecular weight by end group titration=2270g/mole) and goserelin acetate (0.46 g). The acetic acid content of thisamorphous solid was determined by gas chromatography and was found to be0.14%.

[0238] This goserelin-lactide polymer salt (1 g) was added to benzylbenzoate (99%, ex Janssen, 2 ml), and this was heated using a hand heldhot-air gun whilst agitating the mixture until the solid was dissolved.110 μl of this solution formulation contained 3.6 mg of goserelin.

[0239] Formulation 3

[0240] A lactide/glycolide copolymer (95/5) with a single terminalcarboxylic acid (7.86 g, MW=5750, polydispersity=1.50, molecular weightby end group titration 2516 g/mole) and goserelin acetate (0.98 g) weredissolved in glacial acetic acid (100 ml). This solution was frozen byadding dropwise to liquid nitrogen, followed by freeze drying for 2days. The resulting solid was then dried for a further 24 hours at 40°C. The acetic acid content of this freeze dried solid was determined bygas chromatography and was found to be 0.17%.

[0241] This goserelin-lactide/glycolide copolymer mixture (1 g) wasadded to benzyl benzoate ((2 ml, 99%, ex Janssen), and this was heatedusing a hand held hot-air gun whilst agitating the mixture until thesolid was dissolved. 110 μl of this solution formulation contained 3.6mg of goserelin.

[0242] It can therefore be seen that formulation of goserelin as thepolyester salt confers good solubility properties upon the drug, suchthat it can be dissolved in lipophilic solvents such as benzyl benzoatein which goserelin acetate itself is not soluble.

[0243] Biological Evaluation

[0244] Formulations 1-3 were dosed to groups (n=10) of regularly cyclingfemale rats at a dose of 3.6 mg goserelin per rat, as described inExample 16. Following dosing, the animals were found to enter a periodof continuous dioestrus indicating continuous release of goserelin. Theaverage duration of the diostrus period for each group of rats is givenin the following table. From this table it can be seen that all threeformulations gave periods of goserelin release in excess of fourteenweeks. Average duration of dioestrus Formulation No. (days) (±s.e.) 1104 (±5.4) 2  99 (±3.9) 3 101 (±2.8)

[0245] It can further be seen from these examples that the formulationsof the goserelin polyester salt can be provided as solutions which canbe readily administered parentally using a narrow gauge needle, and thatsuch formulations are convenient for the treatment of hormone dependenttumours in man.

EXAMPLE 18

[0246] Formulation 1

[0247] Lactide/glycolide copolymer (9515) with a single terminalcarboxylic acid (4.5 g, Mw=6806, polydispersity=1.55, molecular weightby end group titration=3027 g/mole, inherent vicosity at 1% w/v inchloroform=0.108 dl/g) was dissolved in glacial acetic acid (50 ml). Tothis solution was added goserelin acetate (0.56 g, equivalent to 0.5ggoserelin) and the mixture was stirred for 10 minutes to give a clearcolourless solution. This was frozen by adding dropwise to liquidnitrogen, followed by freeze drying for 2 Days. The resulting solid wasthen dried for a further 24 hours at 40° C. The acetic acid content ofthis freeze dried solid was determined by gas chromatography and wasfound to be 0.3%.

[0248] This goserelin-lactide/glycolide copolymer mixture (1.0 g) wasadded to benzyl benzoate (2.0 ml, 99%, ex Janssen) and was dissolvedwith warming and agitation. The final solution contained 3.67 mg ofgoserelin in 110 μl, and the goserelin content of the final product was10.0% w/w.

[0249] Formulation 2

[0250] The process described above for Formulation 1 was repeated, usinga lactide/glycolide copolymer (95/5) with a single terminal carboxylicacid (4.0 g, Mw=6011, polydispersity=1.56, molecular weight by end grouptitration=2700 g/mole, inherent vicosity at 1% w/v in chloroform 0.099dl/g and 1.12 g of goserelin acetate (equivalent to 1.0 g of goserelin).The acetic acid content of this freeze dried solid was determined by gaschromatography and was found to be 0.83% and the goserelin content ofthe final product was 19.46% w/w.

[0251] This goserelin-lactide/glycolide copolymer mixture (0.54 g) wasadded to benzyl benzoate (2.46 ml, 99%, ex Janssen) and was dissolvedwith warming and agitation. The final solution contained 3.50 mg ofgoserelin in 110 μl.

[0252] Formulation 3

[0253] The process described above for Formulation 2 was repeated, using2.1 g of the lactide/glycolide copolymer and 1.0 g of goserelin acetate(equivalent to 0.9 g of goserelin). The acetic acid content of thisfreeze dried solid was determined by gas chromatography and was found tobe 1.14%, and the goserelin content of the final product was 28.91% w/w.

[0254] This goserelin-lactide/glycolide copolymer mixture (0.36 g) wasadded to benzyl benzoate (2.64 ml, 99%, ex Janssen) and was dissolvedwith warming and agitation. The final solution contained 3.47 mg ofgoserelin in 110 μl.

[0255] Formulation 4

[0256] The process described above for Formulation 1 was repeated, usinga lactide/glycolide copolymer (95/5) with a single terminal carboxylicacid (8.66 g, Mw=5604, polydispersity=1.71, molecular weight by endgroup titration=1960 g/mole, inherent vicosity at 1% w/v inchloroform=0.094 dl/g and 1.08 g of goserelin acetate (equivalent to0.96 g of goserelin). The acetic acid content of this freeze dried solidwas determined by gas chromatography and was found to be 0.08% and thegoserelin content of the final product was 9.90% w/w.

[0257] This goserelin-lactide/glycolide copolymer mixture (1.0 g) wasadded to benzyl benzoate (2.0 ml, 99%, ex Janssen) and was dissolvedwith warming and agitation. The final solution contained 3.67 mg ofgoserelin in 110 μl.

[0258] Biological Evaluation

[0259] Formulations 1-4 were dosed to groups (n=9 or 10) of regularlycycling female rats at a dose of 3.6 mg goserelin per rat, as describedin Example 16. Following dosing, the animals were found to enter aperiod of continuous dioestrus indicating continuous release ofgoserelin. The average duration of the diostrus period for each group ofrats is given in the following table. From this table it can be seenthat all three formulations gave periods of goserelin release for aperiod of about 3 months or more. Average duration of dioestrusFormulation No. (days) (±s.e.) 1 114 ± 1.8 2  94 ± 4.6 3  97 ± 5.3 4  83± 4.3

[0260] It can further be seen from these examples that the formulationsof the drug polyester salt can be provided as solutions which can bereadily administered parentally using a narrow gauge needle, and thatsuch formulations are convenient for treatment of hormone dependenttumours in man.

EXAMPLE 19

[0261] The goserelin-polyester salt (ii) of Example 16 (3.75 g) wasdissolved in dichloromethane (50 ml) which had previously been filteredthrough a 0.45 μm filter. This solution was filtered through a 0.5 μmteflon filter membrane (Whatman WTP) into a flask which had beensterilised using an autoclave. The solvent was removed using a rotaryevaporator to give a viscous liquid, and air was then admitted to therotary evaporator through a 0.5 μm filter. The viscous liquid was warmedand dried under vacuum to give a white foam. The foam obtained wasweighed into autoclaved crimp-top vials in a laminar flow cabinet andfreshly distilled solvents were added to give solution formulations ofthe goserelin-polyester salt which were essentially particulate-free.

[0262] Formulation 1

[0263] 1 g of the solid was added to benzyl benzoate (distilled, bp 106°C. at 0.3 mb, 3 ml) and was warmed using a hot-air gun until dissolved.145 μl of this solution formulation contained 3.6 mg of goserelin.

[0264] Formulation 2

[0265] 1 g of the solid was added to benzyl alcohol (distilled, bp 44°C. at 0.3 mb, 1.7 ml) and was warmed using a hot-air gun untildissolved. 100 μl of this solution formulation contained 3.6 mg ofgoserelin.

[0266] Biological Evaluation.

[0267] Two groups of ten female rats were dosed subcutaneously using a20 gauge needle with formulations 1 and 2 at a dose of 3.6 mg per rat.Terminal blood samples were taken from the rats at subsequent timepoints(1 week (n=4), 4 weeks and 6 weeks (n=3)). The blood samples wereassayed for goserelin by means of radioimmunoassay. Measurable bloodlevels of goserelin were found with both formulations, indicating thatthe solution formulations gave sustained drug release for several weeks.The blood level profile of formulation 1 was found to peak at about fourweeks, whereas with formulation 2 the peak occurred at week one andthereafter the blood levels were found to decline progressively withtime. The blood level profile of formulation 1 is considered to be moredesirable than that of formulation 2 due to the more constant bloodlevels obtained when benzyl benzoate is used as the solvent for thesolution formulation.

[0268] It can further be seen from these examples that the formulationsof the drug polyester salt can be provided as solutions which can bereadily administered parentally using a narrow gauge needle, and thatsuch formulations are-convenient for treatment of hormone dependenttumours in man.

EXAMPLE 20

[0269] A lactide/glycolide copolymer (95/5) with a single terminalcarboxylic acid (9.0 g, Mw=6011, polydispersity=1.56, molecular weightby end group titration=2700 g/mole, inherent vicosity at 1% w/v inchloroform=0.099 dl/g) was dissolved in dichloromethane (100 ml). Tothis was added goserelin acetate (1.124 g, equivalent to 1 g ofgoserelin) with stirring, followed by the addition of methanol (10 ml).The cloudy suspension obtained was stirred at room temperature for aboutone hour until a clear solution was obtained. The solvent was removedusing a rotary evaporator to give a clear viscous liquid. This was thenredissolved in dichloromethane and redried as before. This step wasrepeated twice more, and the viscous liquid finally obtained was driedunder high vacuum to produce a white foam, which was further vacuumdried overnight. The foam was broken to a fine powder which was vacuumdried for one day at room temperature. To this powder was added benzylbenzoate (20 ml, 99%, ex Janssen) and the resultant mixture was gentlywarmed, with agitation, to obtain a solution.

[0270] Biological Evaluation.

[0271] This solution formulation of goserelin was dosed subcutaneouslyusing a 20 gauge needle into each of 45 female rats (220 μl, equivalentto 7.3 mg goserelin). Groups of five rats were terminated and bloodsamples taken at 1 and 4 Days, and 1, 3, 5, 7, 9, 11 and 13 weeks. Inaddition blood samples were taken from the tail vein of groups of fiverats at 2, 4, 6, 8, 10 and 12 weeks. The samples were analysed forgoserelin by means of radioimmunassay, and the results show that thisliquid formulation of goserelin-polyester salt gave measurable bloodlevels of drug for about 11 weeks after dosing and shows that theformulation gives sustained release of goserelin in vivo.

[0272] It can further be seen from these examples that the formulationsof the drug polyester salt can be provided as solutions which can bereadily administered parentally using a narrow gauge needle, and thatsuch formulations would be convenient for treatment of hormone dependenttumours in man.

EXAMPLE 21

[0273] The peptide known as Substance P, in the form of its acetate salt(ex Sigma, 2 mg) was added to dichloromethane (3 ml) and thoroughlyagitated. The peptide showed no indication of dissolving in the solvent,and remained as a cloudy suspension.

[0274] A lactide/glycolide copolymer (70/30) with a single terminalcarboxylic acid (225 mg, Mw=9755, polydispersity=1.52, molecular weightby end group titration=1800), was added to dichloromethane (25 ml). Thiswas stirred for 15 minutes to give a clear colourless solution. To thiswas added a solution of Substance P (25 mg) in methanol (0.5 ml). Theresulting cloudy suspension was stirred for 1 hour, by which time acompletely clear solution had formed. The solvent was removed by rotaryevaporation and the clear ‘glassy’ solid obtained was redissolved indichloromethane (5 ml) and reevaporated. This was repeated twice. Thefinal solid was dissolved in dichloromethane (3 ml) and the solution wasdropped slowly onto PTFE coated cloth, allowing the solvent to evaporateto form a thin film of a clear colourless glassy solid (peptide content9.1% w/w).

[0275] This film (96.8 mg) was placed in a small vial and phosphatebuffered saline (2 ml, pH 7.4) was added (buffer was previously filteredthrough a 0.2 μm filter and contained 0.02% sodium azide as apreservative). The vial was placed in an incubator at 37° C. and thebuffer was removed and replaced periodically. The buffer which wasremoved was analysed for release of Substance P, using an ultravioletspectrophotometer (Hewlett Packard 8452A) at 210 nm, against standardsolutions of substance P. The results show that Substance P can bedissolved in dichloromethane when formed as the salt of acarboxy-terminated lactide/glycolide copolymer, and can be processed inthis solvent to give a thin film, which gives continuous release of thepeptide for a period of about 4 weeks.

EXAMPLE 22

[0276] An aqueous solution of leuprolide acetate (otherwise known asleuprorelin acetate), (300 μl of a 330 mg/ml solution) is added underhigh shear conditions, to 20 ml of a 10% w/w solution ofpoly(hydroxystearic acid) having a number average molecular weight ofabout 2000, in Miglyol 812 (triglycerides of medium chain saturatedfatty acids including linolenic acid, ex Dynamit Nobel, UK), to form theleuprolide-polymer salt, in part, at the oil/aqueous interface, whichsalt stabilises the resultant water-in-oil colloidal suspension. Thewater is removed at 50° C. by stirring under high vacuum until themixture no longer froths and bubbles, to give an oily composition whichexhibits a very faint haze, and which is suitable for oraladministration.

EXAMPLE 23

[0277] Lys⁸-vasopressin acetate salt (2 mg, ex Sigma) was added todichloromethane (3 ml) and agitated. The peptide showed no indication ofdissolving in the solvent and remained as a cloudy suspension.

[0278] A lactide/glycolide copolymer (70/30) with a single terminalcarboxylic acid (225 mg, Mw=9755, polydispersity=1.52, molecular weightby end group titration=1800), was added to dichloromethane (5 ml). Thismixture was stirred for 15 minutes to give a clear colourless solution.To this was added Lys⁸-vasopressin (25 mg, ex Sigma) and methanol (0.5ml). The resulting cloudy suspension was stirred for 1 hour, by whichtime a completely clear solution had formed. The solvent was removed byrotary evaporator and the clear ‘glassy’ solid obtained was redissolvedin dichloromethane (5 ml) and re-evaporated. This was repeated twice.The final solid was dissolved in dichloromethane (3 ml) and the solutionwas dropped slowly onto PTFE coated cloth, allowing the solvent toevaporate to form a thin film of a clear colourless glassy solid (Lys-vasopressin content 10% w/w).

[0279] This film (97.31 mg) was placed in a small vial and phosphatebuffered saline (2 ml, pH 7.4) was added (buffer was previously filteredthrough a 0.2 μm filter and contained 0.02% sodium azide as apreservative). The vial was placed in an incubator at 37° C. and thebuffer was removed and replaced periodically. The buffer was analysedfor release of Lys⁸-vasopressin using an ultraviolet spectrophotometer(Hewlett Packard 8452A) at 210 nm against standard solutions ofLys⁸-vasopressin. The results of this test are shown in the followingtable. The experiment shows that Lys⁸-vasopressin can be dissolved indichlormethane, when formed as the salt of a carboxy-terminatedlactide/glycolide copolymer, and that the resulting mixture givescontinuous release of the peptide for a period of at least four weeks.Release of Lys⁸-vasopressin in vitro Release of Lys⁸-vasopressin Time(days) from film (%) 1 4.11 4 5.45 7 5.55 14 5.75 21 26.82 28 47.27

EXAMPLE 24

[0280] Two formulations of ZENECA ZD6003 ([Het⁻¹, Arg¹¹,Ser^(17,27,60,65)] human G-CSF (granulocyte-colony stimulating factor)modified with polyethylene glycol 5000 as described in Reference Example4 or 7 of European Patent Publication No. 0 473 268) inlactide/glycolide copolymer as follows.

[0281] (i) Dichloromethane (4 ml) was added to a freeze-driedpreparation of ZD6003 (39.72 mg). This resulted in an opaque dispersionof drug in the solvent. A lactide/glycolide copolymer (75/25) with asingle terminal carboxylic acid (363.6 mg, Hw 9963, polydispersity=2.19,molecular weight by end group titration=2815) was added, and a clearsolution formed.

[0282] This solution was added to a solution (400 ml) of methylcellulose (0.25% w/v Methhocel, 15 mPa.s, ex Fluka) in water under shear(2150 RPM, Heidolph RZR50 stirrer). After stirring at this rate for 3minutes the stirring speed was reduced to 800 RPM. The resultingparticles were then allowed to settle under gravity for 30 minutes,whilst keeping the solution cool over ice. The supernatant was thendiscarded and the particles were washed by resuspending in ice-colddistilled water (50 ml), and centrifugation at 1000 RPM. This wasrepeated four times and the particles were then finally freeze dried.

[0283] Particles made in this way were of good quality, being sphericaland of a mean size of 32 μm as determined by image analysis from opticalmicroscopy. The drug content of these particles was determined byextraction followed by HPLC analysis and was found to be 9.45%,representing an incorporation efficiency of 96% of the drug used to formthe microparticles.

[0284] (ii) Dichloromethane (4 ml) was added to a freeze-driedpreparation of ZD6003 (44.18 mg). This resulted in an opaque dispersionof drug in solvent. A lactide/glycolide copolymer (75/25, 364.1 mg,Mw=16,800 by size exclusion chromatography, polydispersity=2.2, exBoehringer Ingelheim) was added. An attempt to determine the molecularweight of the polymer by end group titration was performed, but was notpossible due to very low levels of titratable moieties, and consequentlythis polymer does not have a terminal carboxylic acid. The mixture ofthe drug solution and the polymer did not become clear upon addition ofthe polymer and the mixture remained as a turbid dispersion, indicatingthat, as expected, in the absence of acid end groups in the polymer, nopeptide-polyester salt could form.

[0285] This mixture was added to a solution (400 ml) of methyl cellulose(0.25% w/v Methocel, 15 mPa.s, Fluka) in water under shear (2150 RPM,Heidolph RZR50 stirrer). After stirring at this rate for three minutesthe stirring speed was reduced to 800 RPM. The resulting particles werethen allowed to settle under gravity for 30 minutes, whilst keeping thesolution cool over ice. The supernatant was then discarded and theparticles were washed by resuspending in distilled water (50 ml) andcentrifugation at 1000 RPM. This was repeated four times and theparticles were then finally freeze dried.

[0286] Particles made in this way were of inferior quality, comparedwith those obtained in (i) above, with some being of irregular shape andof a mean size of 40 μm as determined by image analysis from opticalmicroscopy. The drug content of these particles was determined byextraction followed by HPLC analysis and was found to be 2.05%,representing an incorporation efficiency of 19% of the drug used to formthe microparticles.

[0287] The above example shows that ZD6003 can be dissolved indichloromethane when in the presence of a polymer with a single terminalcarboxylic acid, despite dichloromethane iself being a non-solvent forthe drug. In addition such a solution can be used to form microparticlesof drug and polymer with a very high rate of incorporation of drug. Incontrast, the above example also shows that ZD6003 cannot be dissolvedin dichloromethane in the presence of a polymer, when such a polymerdoes not have a terminal carboxylic acid, and forms only a hazydispersion. Furthermore such hazy dispersions of ZD6003 in a solution ofpolymer with no terminal carboxylic acid result in poor incorporation ofdrug when processed to form microparticles.

EXAMPLE 25

[0288] (i) Goserelin acetate (22.47 mg, equivalent to 19.99 mggoserelin) was added to benzyl benzoate (2.21 g, 99%, ex Janssen). Thismixture was placed in an incubator at 40° C. and was stirredcontinuously for 9 Days using a magnetic stirrer. After 2 and 9 Daysaliquots were taken and centrifuged for 15 minutes at 13,000 RPM topellet undissolved drug. Aliquots of supernatant (approx. 100 mg) wereweighed accurately into 50 ml volumetric flasks. To each was addedglacial acetic acid (2 ml), followed by making up to volume with-anaqueous solution of trifluoroacetic acid (0.5% v/v). A portion of thissolution was placed in a centrifuge tube and was centrifuged at 13,000RPM for 15 minutes to separate suspended material. The supernatant wasthen assayed for goserelin content, using HPLC. No goserelin wasdetectable in either sample. The limit of detection of goserelin in thisHPLC assay was 0.2 μg/ml and the limit of quantification was 0.5 μg/ml.Thus the equilibrium solubility (at 40° C.) of goserelin in benzylbenzoate can be estimated from the above as less than 0.2 μg/mg.

[0289] (ii) A lactide/glycolide copolymer (95/5) with a single terminalcarboxylic acid (291.9 mg, Mw=6742, polydispersity=1.61, molecularweight by end group titration=2565 gm/mole, inherent vicosity at 1% w/vin chloroform=0.103 dl/g) was added to benzyl benzoate (3.38 g, 99%, exJanssen) to form a solution. To this was added goserelin acetate (22.52mg, equivalent to 20.03 mg goserelin). This mixture was incubated andsampled as described in (i) above. No goserelin was detectable in thebenzyl benzoate at 2 Days, but at 9 Days a level of approximately 0.2 μggoserelin per mg of benzyl benzoate was detected. The limit of detectionof goserelin in this HPLC assay was as indicated in (1) above. From thisit can be shown that the equilibrium solubility (at 40° C.) of goserelinin benzyl benzoate, when present as a simple mixture with alactide/glycolide copolymer, can be estimated as 0.2-0.5 μg/mg.

[0290] (iii) A lactide/glycolide copolymer (95/5) with a single terminalcarboxylic acid (9.0 g, Mw=6011, polydispersity=1.56, molecular weightby end group titration=2700 g/mole, inherent vicosity at 1% w/v inchloroform=0.099 dl/g) was dissolved in dichloromethane (100 ml). Tothis was added goserelin acetate (1.124 g, equivalent to 1 g goserelin)with stirring, followed by the addition of methanol (10 ml). The cloudysuspension obtained was stirred at room temperature for about 1 houruntil a clear solution was obtained. The solvent was removed using arotary evaporator to give a clear viscous liquid. This was thenredissolved in dichloromethane and redried as before. This step was thenrepeated twice more and the viscous liquid finally obtained was driedunder high vacuum to produce a white foam, which was further vacuumdried overnight. The foam was broken to a fine powder which was vacuumdried for 1 Day at room temperature. To this powder was added benzylbenzoate (20 ml, 99%, ex Janssen) and the resultant mixture was gentlywarmed, with agitation, to obtain a solution.

[0291] The solution was thoroughly mixed and a 1 ml sample was placed ina centrifuge and spun at 14,000 RPM for 30 minutes. An aliquot of thesupernatant was carefully removed and weighed into a 50 ml volumetricflask. The sample was assayed for goserelin content as described in (i).The goserelin content of this solution was found to be 24.6 pg/mg benzylbenzoate.

[0292] This example shows that benzyl benzoate is a very poor solventfor goserelin acetate. Furthermore, the addition of a lactide/glycolidepolymer to form a simple mixture with goserelin acetate in benzylbenzoate does not lead to a marked increase in the equilibriumsolubility of goserelin acetate in benzyl benzoate. However,goserelin/polyester salt could be dissolved in benzyl benzoate to form asolution containing goserelin at a concentration very much higher thanthe estimated equilibrium solubility of free goserelin in this solvent.

1. A composition containing or comprising, as initially made, a saltformed from a cation derived from a peptide containing at least onebasic group and an anion derived from a carboxy-terminated polyester;the composition being in the form of a solution or dispersion of thesalt in a solvent which is a solvent for the free polyester but not asolvent for the free peptide, the particle size of the salt in saiddispersion being less than 5 μm and preferably less than 0.2 μm; or inthe form of microparticles or an implant, for injection or sub-dermalimplantation.
 2. A composition as claimed in claim 1 wherein the peptideis pharmacologically active, and is selected from oxytocin, vasopressin,adrenocorticotrophic hormone (ACTH), epidermal growth factor (EGF),prolactin, luteinising hormone, follicle stimulating hormone, luliberinor luteinizing hormone releasing hormone (LHRH), insulin, somatostatin,glucagon, interferon, gastrin, tetragastrin, pentagastrin, urogastrone,secretin, calcitonin, enkephalins, endorphins, kyotorphin, taftsin,thymopoietin, thymosin, thymostimulin, thymic humoral factor, serumthymic factor, tumour necrosis factor, colony stimulating factors,motilin, bombesin, dinorphin, neurotensin, cerulein, bradykinin,urokinase, kallikrein, substance P analogues and antagonists,angiotensin II, nerve growth factor, blood coagulation factor VII andIX, lysozyme chloride, renin, bradykinin, tyrocidin, gramicidines,growth hormones, melanocyte stimulating hormone, thyroid hormonereleasing hormone, thyroid stimulating hormone, parathyroid hormone,pancreozymin, cholecystokinin, human placental lactogen, human chorionicgonadotrophin, protein synthesis stimulating peptide, gastric inhibitorypeptide, vasoactive intestinal peptide, platelet derived growth factor,growth hormone releasing factor, bone morphogenic protein, and syntheticanalogues and modifications and pharmacologically-active fragmentsthereof.
 3. A composition as claimed in claim 1 wherein the peptide ispharmacologically inactive and is selected from polyarginine, polylysineand poly(arginine-co-lysine), (co-)polymers of neutral amino acids, inD-, L- or DL-form, with arginine and/or lysine in D-, L- or racemicform, or peptides or (co-)polypeptides in which the peptide chains areterminated in whole or in part by a basic group at the N-terminus andthe backbone is comprised of neutral amino acid residues.
 4. Acomposition as claimed in claim 1 wherein the polyester is selected fromthose derived from hydroxy-acids and those derived from thepolycondensation of diols and/or polyols with dicarboxylic acids and/orpolycarboxylic acids.
 5. A process for the manufacture of a solution ordispersion of a salt as claimed in claim 1, which comprises (a)dissolving the peptide containing at least one basic amino acid, in freebase form or in the form of a salt with a weak acid and thecarboxy-terminated polyester in a neutral, polar solvent in which bothare soluble, removing the solvent or most of the solvent, and adding theremaining concentrated solution to an excess of a non-solvent for thepeptide-polyester salt, or (b) dissolving the peptide containing atleast one basic amino acid, in free base form or in the form of a saltwith a weak acid, and the carboxy-terminated polyester, in a solvent inwhich both are soluble, and which is capable of being removed byfreeze-drying, freezing the resulting solution at high speed,freeze-drying the resulting frozen mixture, dispersing the resultingmixture in a solvent for the polyester component, and allowing themixture to dissolve as the peptide-polyester salt is formed, or (c)reacting the peptide, containing at least one basic amino acid, in theform of a salt with a strong acid, with a polyester wherein some or allof the polyester is in the form of a carboxylic acid salt with asuitable alkali metal or alkaline earth metal.
 6. A composition asclaimed in claim 1, comprising a pharmacologically active peptide and apolyester, for extended release of the peptide drug, characterised inthat the composition is in the form of microparticles from 0.2 μm to 500μm in diameter, suspended in a pharmaceutically acceptable injectionvehicle.
 7. A composition as claimed in claim 6 wherein the injectionvehicle is aqueous or is an organic vehicle which is a non-solvent forthe materials used, or, for highly lipophilic polyesters, is ahydrophilic organic injection vehicle.
 8. A composition as claimed inclaim 1, comprising a pharmacologically active peptide and a polyester,for extended release of the peptide drug, characterized in that thecomposition is in the form of a pharmaceutically acceptable solution,comprising: (a) a peptide drug, containing at least one basic aminoacid, as hereinbefore defined, having a molecular weight of at least 300Da, which is in the form of a salt with the polyester, the saltcomprising a cation of the basic peptide and an anion of acarboxy-terminated polyester, (b) a solvent which is a solvent for thefree polyester but not a solvent for the free peptide, (c) an excess ofthe polyester, and optionally (d) some of said peptide in solubilised orcolloidally dispersed free form.
 9. A composition as claimed in claim 8wherein the basic peptide drug is a synthetic analogue of luteinisinghormone releasing hormone, selected from buserelin ([D-Ser(Bu^(t))⁶,des-Gly-NH₂ ¹⁰]-LHRH(1-9)NHEt), deslorelin ([D-Trp⁶, des-Gly-NH₂¹⁰]-LHRH(1-9)NHEt), fertirelin ([des-Gly-NH₂ ¹⁰]-LHRH(1-9)NHEt),goserelin ([D-Ser(Bu^(t))⁶, Azgly¹⁰]-LHRH), histrelin ([D-His(Bzl)⁶,des-Gly-NH₂ ¹⁰]-LHRH(1-9)NHEt), leuprorelin ([D-Leu⁶, des-Gly-NH₂¹⁰]-LHRH(1-9)NHEt), lutrelin ([D-Trp⁶, MeLeu⁷, des-Gly-NH₂¹⁰]-LHRH(1-9)NHEt), nafarelin ([D-Nal⁶]-LHRH), tryptorelin([D-Trp]-LHRH), and pharmacologically active salts thereof.
 10. Acomposition as claimed in claim 8 wherein the solvent is selected frombenzyl benzoate, benzyl alcohol, ethyl lactate, glyceryl triacetate,esters of citric acid, and low molecular weight (<1000) polyethyleneglycols, alkoxypolyethylene glycols and polyethylene glycol acetates.11. A composition as claimed in claim 8 wherein the ratio of basicpeptide drug-polyester salt to free polyester is from 1:0 to 0.1:10. 12.A composition as claimed in claim 8 wherein the ratio of total solids tosolvent is from 2% w/v to 40% w/v.
 13. A process for the manufacture ofa pharmaceutical composition as claimed in claim 8 which comprises (a)dissolving an intimate mixture of the peptide drug and the polyester inthe pharmaceutically acceptable solvent; or (b) slowly adding a solutionof the peptide drug in a 1-6C alkanol to a solution of the polyester ina solvent suitable for injection, whereafter, if the solvent in thestarting peptide solution is not pharmaceutically acceptable forinjection, it is removed.